JPH0727765A - Assay using absorption material - Google Patents

Abstract

PURPOSE: To carry out dynamic assay so as to make a coupling agent into contact an object to be analyzed, by directly applying a sample containing the object to be analyzed, to the absorbent supporting at least one coupling agent for the object to be analyzed. CONSTITUTION: A support 30 made of an absorbent is divided into a plurality of test zones 32, and each of the zones 32 has an outer surface 34. A coupling agent spot or a trapping area for a coupling agent for an object to be analyzed and also the object to be analyzed itself as necessary is formed on the outer surface 34. Further, the coupling agent and the objective spot can be set on the outer surface 34. The coupling agent and the object to be analyzed are directly coupled through reception or covalent bond, or and can be coupled with latex on the outer surface 34. The degree of porosity of the absorbent for forming the test zones 32 prevents a sample from crosswise flowing from one of the test zones to another one of the zones. Further, the test zones 32 are separated from one another by a plurality of passages 40 cut in the absorbent.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This application relates to a method for performing an assay, in particular a so-called kinetic assay or a "flow" assay. More specifically, the present application relates to performing assays in which a binding agent and an analyte are contacted on an absorbent material.

[0002]

BACKGROUND OF THE INVENTION Using a device having an absorbent material or zone that provides a flow of an analyte-containing liquid through a membrane and supports antigens, antibodies or other types of binders for analytes. Performing immunological assays by is known in the art.

US Pat. No. 4,3, issued to Tom et al.
66,241 disclose flow assay devices and assays using such devices. The assay device has an immunological zone to which one member of the immunological pair is immobilized. The liquid absorption area that draws the liquid sample from the immunoadsorption area is located near the immunoadsorption area. This absorption zone can control the rate at which the liquid sample is drawn from the immunoadsorption zone.

US Pat. No. 4,632,901 issued to Valkirs et al. Discloses an apparatus and method for performing an immunoassay, in which an antibody such as a monoclonal antibody is attached to a membrane or filter. . The absorbent material is located below the membrane or filter, which directs the flow of liquid sample through the membrane or filter. The analyte in the liquid sample binds to the antibody on the membrane or filter. A labeled antibody to the analyte can then be added. Then, the unbound labeled antibody is removed by a washing step. The presence of labeled antibody on the membrane or filter after the wash step indicates the presence of analyte in the sample being assayed.

[0005]

[Problems to be Solved by the Invention and Means for Solving the Problems]
According to a feature of the invention, at least one for the analyte
An analyte assay is provided that applies an analyte-containing sample directly to an absorbent material on which one binder is supported.
Thus, in this assay, the sample containing the analyte was supported on at least a portion of the surface of the absorbent material,
Applied to the surface of an absorbent material having at least one binder for the analyte. The absorbent material used in the assay is one that is capable of inducing a capillary flow, thereby drawing the liquid applied to the surface of the absorbent material into the absorbent material. In addition, the absorbent material is characterized by porosity or density, which allows the absorbent material to retain on its surface uncharged particles having a size of at least 0.1 microns and not more than 10 microns. The sample flows through the supported binder into the absorbent material and the analyte binds with the binder. The analyte bound to the supported binder is then measured. The sample passes through the binder and flows through the absorbent material by capillary type movement by the absorbent material. The absorbent material can be contained within a container and the container can be made of plastic. The types of assays intended to be included within the scope of the present invention are known to those of ordinary skill in the art. Such assays include competitive assays,
Included are sandwich assays, enzyme linked immunosorbent (ELISA) assays, agglutination assays, indirect assays and other assays in the art.

In accordance with another feature of the present invention, a device is provided that comprises an absorbent material partitioned into a plurality of test areas. Each of the plurality of test areas has at least 0.1
It has a surface capable of holding uncharged particles having a size in microns of no more than 10 microns. Such devices can be used in performing multiple assays of the type described above. The absorbent material of the device minimizes sample cross-flow between the test areas, thus allowing the sample to be applied to each of the test areas.

In one embodiment, the device comprises a cover having a plurality of openings, each opening being located above the surface of the corresponding test area. The preferred embodiment of the cover is made of a pressure sensitive material such as vinyl and has die-cut openings.

According to another feature of the present invention, there is provided a device comprising the absorbent material described above. The surface of the absorbent material has at least one well portion, each of the at least one well portion defining a test area. At least one well portion can receive the sample. Preferably, the surface of the absorbent material has a plurality of well portions. More preferably, the surface of the absorbent material further comprises at least one groove, each of the at least one groove being located between two of the plurality of wells. At least one groove is employed to catch the overflow of sample applied to the well.

Referring now to the drawings, the solid support according to the present invention can be in the form of a cylinder 10 having a top surface 12. The cylindrical support 10 can be contained in a container consisting of a bottom portion 18 and a top lid portion 20. The lid 20 of the container is the surface 12 of the cylinder 10.
Has an opening 22 surrounding the.

The embodiment shown in FIG. 1 illustrates an embodiment according to the present invention known as a plus "+" or minus "-" capture assay. The analyte area 14 and the binder area 16 are located on the surface 12 of the cylinder 10 in one embodiment. The analyte and binding agent can be bound directly or covalently to surface 12 or can be bound to latex particles on surface 12. It is understood that in this embodiment, analyte area 14 and binder area 16 intersect or overlap each other, resulting in the shape of a plus "+" sign. When an assay is to be performed according to this embodiment, the analyte is placed in area 14 of surface 12 and the binding agent for the analyte in area 16. When the surface 12 is contacted with a sample suspected of containing the analyte, the analyte, if present, in the area 1
It binds with the binder of 6. After adding the sample, the surface 12 is contacted with a tracer having a detectable label that binds the analyte. If the analyte is present in the sample,
Tracers with detectable labels are in areas 14 and 1
Combine with both 6. After subsequent washing of the surface 12, a plus "+" sign appears on the surface 12 of the support 10, thus indicating the presence of the analyte. If the analyte is not present in the test sample, the tracer with the detectable label will only bind to the analyte in area 14. As a result, this test result reads as a minus "-" sign, thus indicating the absence of antibody to the capture antigen in the sample.

Alternatively, as shown in FIG. 4, a binding agent spot 42 containing a binding agent for the analyte and a control spot 44 containing a non-reactive antigen or antibody (eg gamma-globulin) are provided on the surface 12. Can be placed on top. The non-reactive antigen or antibody contained in the control spot 44 serves to detect contaminants by capturing non-specific substances. If non-specifically bound contaminants are present, such contaminants bind to the non-reactive or non-specific antibody contained in control spot 44 and cause control spot 44 to be expressed. When the sample is applied to the surface 12, the analyte, if present, will bind to the binder in binder spot 42 and cause binder spot 42 to develop. If no analyte is present, binder spot 42 will not develop. The binder can be bound directly to the surface 12 or to the latex particles on the surface 12. When latex particles are used, binder spot 42 contains latex particles with bound binder, and control spot 44 is latex particles with non-reactive (ie non-specific) antigen or antibody bound. Contains. The sample containing the analyte is surface 12
The analyte binds to the latex particles contained in binder spot 42 and not to the particles contained in control spot 44. If analyte is not present in the sample, neither binder spot 42 nor control spot 44 will be expressed. If the control spot 44 is expressed, regardless of whether the binder spot 42 is expressed, the expression of the control spot 44 indicates the presence of contaminants in the sample and / or the test material. Control spot 4
4 is actually expressed by the application of the test sample,
The test result should be ignored and the presence of the analyte should be measured again using another support.

In another embodiment, as shown in FIG. 3, the support 30 made of absorbent material as described above is divided into a plurality of test areas 32. Each of the test areas 32 has a surface 34 on which the binding agent for the analyte and, if desired, the analyte are also arranged as described above to bind the binding agent spot or "capture" area. Can be formed. The binder and control spots described above can also be placed on the surface 34. The binding agent and, if desired, the analyte can be bound directly or covalently to the surface 34, or to latex particles on the surface 34. Test area 32
The porosity of the absorbent making up is sufficient to minimize lateral flow of sample from one test zone to another.
The test areas 32 are separated from each other by a plurality of passages 40 cut into the absorbent material to further inhibit cross flow between the test areas 32. Thus, this embodiment makes it possible to carry out multiple assays using a device with multiple test zones, whereby the sample can be applied to multiple test zones, whereby one test can be performed. Samples applied to an area do not cross flow or contaminate adjacent test areas.

A cover 36 having a plurality of openings 38 may be disposed on top of the support 30. Each of the openings 38 is located above the surface 34 of the corresponding test area 32. In the preferred embodiment, the cover 36 is made of pressure sensitive vinyl with die-cut openings.

In yet another embodiment, as shown in FIGS. 5 and 6, the support 50 is shown in the form of a microtiter plate having an absorbent substrate 52 as described above, the surface 53 of which has a plurality of wells. 54 are cut out, each of the wells 54 defining a test area. Well 54
Are bounded by circular walls 55. However, it should be understood that the shape of the well 54 is not limited to a circular shape. A plurality of supports 50 may be included in the tray 60.

Each well 54 has a surface 56 to which a sample is applied. A binding agent for the analyte and, if desired, the analyte are also disposed on surface 56. Binder and analyte can be present in the form of analyte area 14 'and binder area 16'. The surface 56 is
Alternatively, one can have a binder spot and a control spot as described above. The binder and, if desired, the analyte can be passively or covalently bound to surface 56 or to latex particles on surface 56. Disposed between the wells 54 are passageways 58, each of which is bounded by walls 57, 59, 61 and 63. Each of the channels 58 receives an overflow of sample applied to the adjacent well 54. Thus, the passages 58 serve to provide space between adjacent wells 54 as well as prevent lateral flow of sample between adjacent wells 54.

In a competitive assay, the analyte and tracer compete with the binding agent specific for the analyte and tracer. A tracer is an analyte or suitable analog thereof that is linked to a detectable label or marker. The tracer and the analyte compete for a finite number of binding sites on the binder, and the amount of tracer bound to the binder is inversely proportional to the amount of analyte in the sample. The amount of tracer and the amount of analyte can be determined by measuring the amount of label present. Labels or markers that are part of the tracer include, for example, radioisotopes of iodine, cobalt or tritium, enzymes, fluorescent dyes,
Absorbing dye, chemiluminescent substance, spin label, biotin,
It can be a detectable marker such as colored particles or any other labeling substance known to one of ordinary skill in the art. A preferred label consists of colored particles such as colloidal gold.

When using the sandwich assay, the analyte-specific binding agent is contacted with a sample containing or suspected of containing the analyte. The analyte present in the sample binds the binding agent. After the sample has passed through the binder and into the absorbent material, the analyte-binder complex is contacted with the tracer. A tracer is a ligand specific to the analyte to be assayed. For example,
The tracer can be an antibody that is 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 the indirect sandwich assay, the analyte bound to the supported binding agent is contacted with the binding agent for the analyte that becomes bound to the analyte bound to the supported binding agent. The tracer used in this assay is a labeled ligand that binds the binding agent bound to the analyte bound to the supported binding agent.

In the ELIZA 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. The ELISA method can be performed as a sandwich assay or a competitive assay.

In the agglutination assay, a binding agent consisting of particles sensitized with an antigen or antibody specific for the analyte is contacted with a sample suspected of containing the analyte. The presence of the analyte is evidenced by the agglomeration of solid particles.

The binding agent used in the assay according to the present invention depends on the analyte to be assayed. For example, where the analyte is an antigen or hapten, the binding agent can be an antibody specific for the analyte or a naturally occurring substance. When the analyte is an antibody, the binding agent can be an antibody specific for the analyte, an antigen or a naturally occurring substance.

The assay can be used to measure a wide variety of analytes. Examples of analytes that can be assayed according to the present invention include drugs, hormones, macromolecules, antibodies, microorganisms, toxins, polypeptides, proteins, polysaccharides, nucleic acids and the like. Selection of the appropriate analyte is believed to be within the skill of those in the art.

The absorbent material used in the assay according to the present invention can be any absorbent material having a porosity such that it can hold uncharged particles of at least 0.1 micron and no more than 10 microns. The absorbent material is wettable (hydrophilic) and provides a capillary movement of liquid through the absorbent. Although the absorbent material can retain uncharged particles of at least 0.1 micron and no greater than 10 microns, assays can be performed using the absorbent material of the present invention without the use of uncharged particles.

The absorbent material used must properly regulate the flow of sample through the absorbent. Flow regulation is important to control the interaction of binder and specimen on the absorbent surface. Fast flow results in loss of sensitivity because the analyte and binder do not have sufficient reaction time. If the flow through the surface and binder is too slow, nonspecific components in the sample may not be properly separated from the binder, which can result in strong nonspecific reactions. Thus, properly regulated flow increases the sensitivity of the assay. The absorbent also preferably prevents "backflow" of the sample onto the absorbent surface. Further, when the absorbent is divided into multiple test areas that perform multiple assays, the absorbent prevents or minimizes sample lateral flow between the test areas, thereby allowing one test area with the sample to move to another. Prevent the sample from contaminating the test area.
When the absorbent is divided into multiple test zones, too slow sample flow can cause cross flow of sample between the test zones in addition to increasing non-specific reaction. The absorbents according to the invention, most preferably those having an upper limit of porosity in the above range, allow the sample to flow downwardly and rapidly through the absorbent so as to prevent cross flow, in which case an assay is required. The velocity of such a flow is not excessive so that it has sufficient sensitivity. The absorbent can be placed in a plastic or fiberboard container or container.

In a preferred embodiment, the absorbent is a non-fibrous material, in particular an absorbent porous plastic which itself is "wettable" and capable of providing a capillary flow into the plastic. Examples of non-fibrous plastic absorbent materials that can be used are polyolefins, polyesters, porous polyvinyl chloride and polystyrene.
The porous plastic absorbent has the porosity or density as described above. The porous plastic is in a preferred embodiment a suitable material such as a cellulosic material that provides the required porosity by filling the pores of the plastic so that the porous material provides the required pore size as described above. Filled with various materials. The cellulosic material does not adversely affect the absorbency and wettability of the plastic. A preferred cellulosic material is acetyl cellulose. A preferred absorbent material is a porous polyethylene absorbent having a surface with pores that have been compressed using a cellulosic material. Such a support is available from Porex Technologies Corporation of Georgia.
S. Corp.) as a MEMPOR ™ porous plastic matrix support system.

The porous plastic may be a hydrophobic porous plastic which becomes hydrophilic or wettable by the addition of a wetting agent such as a surfactant. The surfactant can be applied to all or part of the porous plastic material (eg, after saturating the porous plastic support with the cellulosic material). A plastic that is hydrophobic is contacted with a surfactant to render all and / or a portion of the porous plastic surface hydrophilic or wettable. The binder used in this assay is applied to the "wettable" portion of the porous material. If insufficient amounts of detergent are applied, the flow rate of sample and labeled antibody to the absorbent is slow, thus affecting the sensitivity of the assay.

Surfactants can be applied to the entire surface or part of the surface of the support so that either the entire surface or part of the surface becomes hydrophilic.

When the surfactant is carefully applied to the area where the binder is to be placed, the sample and tracer are forced to flow in the most hydrophilic or wetted area where the binder is placed. This enhances the immunoconcentration mechanism and minimizes sample flow across the entire surface of the absorbent.

In another embodiment, the absorbent can be in the form of compressed fiber discs. The compressed fibers can be hydrophilic or wettable and can provide a capillary flow into the fiber disc. The fiber disc also has a porosity or density that allows the disc to hold uncharged particles having a size of at least 0.1 micron and no greater than 10 microns. The fibers are compressed to produce the required porosity or density described above. The surface of the absorbent material bearing the binder is preferably a smooth surface so as to enhance the ability to read the tracer on said surface.

The surface of the absorbent material may be coated or treated with a material such as BSA or any other inhibitor known in the art to prevent non-specific absorption.

Compressed fiber discs can be made by slurrying the fibers and mixing the fibers with the appropriate inhibitory protein (s), buffer and detergent. A preferred slurry consists of polyester and acetyl cellulose. The slurry is poured into a paper compactor. Excess aqueous material is removed during the compression process. The compression is performed by compressing the disk and at least 0.
One micron is operated to provide sufficient disk density or porosity to hold uncharged particles no greater than 10 microns. The compressor has a fine mesh screen that smoothes the surface of the disc. The smoothness of the disc provides the readability of the assay. In a preferred embodiment, the disk has fibers on its surface that trap latex particles, microspheres or other uncharged particles. The fibers also direct the flow of sample, tracer and / or reagent downwards through the capture or binder zone rather than radially from the capture zone. This allows the assay to achieve maximum sensitivity.

According to another embodiment, the absorbent may be made of a porous ceramic material. The ceramic absorbent can be defined as a non-fibrous, inorganic porous matrix. The ceramic absorbent should be at least 0.1 micron of ceramic absorbent throughout the matrix.
It has a uniform or identical porosity or density capable of holding uncharged particles having a size not exceeding 0 micron. The ceramic absorbent can have a pore size that is preferably from about 0.5 micron to about 10 microns. Preferably, the ceramic absorbent is about 10% to about 8%.
It has a uniform porosity of up to 0%, most preferably about 40%.

The porous ceramic absorbent is microporous throughout and there are no macropores. The porous ceramic absorbent is, in a preferred embodiment, a unitary structure molded as one piece. The absorbent can be made from an aluminum oxide containing material such as alumina.

The porous ceramic sorbent can be made from a feedstock containing alumina in a purity range of about 50% to about 99.9%. Alumina is ground to a uniform particle size and then spray dried. Following spray drying, the uniform alumina is mixed with a binder, release agent and / or particulates to produce a uniform powder. Suitable particulates are those that break up upon combustion of the ceramic, leaving pores in the ceramic material. Examples of such fine particles include latex fine particles. The powder can then be processed in a number of ways to produce raw dough. Examples of such processes include extrusion into rods, rotary compression into sheets, or pill compression to form any design, or injection molding to form any design. Preferably the powder is pill pressed.

The shape of the absorbent can vary depending on the design of the mold used in the pill compaction operation. The composition of the alumina, the binder, the amount of material used and the compression pressure affect the porosity of the final product. The higher the compression pressure and / or the combustion temperature, the lower the porosity of the absorbent. Raw dough is burned at a temperature that is effective to harden the raw dough. The preferred combustion temperature is about 1,000 ° C to about 1.5
Up to 00 ° C. During the combustion process, binders, release agents and particulates evaporate from the alumina matrix, thereby creating pores in the hardened ceramic. The combustion temperature affects the shrinkage of the absorbent and the porosity of the final ceramic absorbent product. After combustion, the ceramic absorbent can be evaluated for porosity using flow rate analysis, water saturation testing and mercury insertion porosity testing. It should be understood that the contact time between the sample and the ceramic absorbent surface depends on the pore size of the absorbent. Larger pore size of the absorbent results in faster sample flow through the absorbent and shorter contact time between the sample and the absorbent surface, while smaller pore size results in less sample flow through the absorbent. The flow becomes slower and the contact time between the sample and the absorbent surface is longer. To provide optimum contact time between the sample and the absorbent surface, the ceramic absorbent preferably has a pore size of from about 0.5 micron to about 10 microns, as described above. The flow rate can also be adjusted to give different assay conditions and contact times by varying the compression pressure and / or the combustion temperature.

According to yet another feature of the present invention,
The absorbent material has a large number of fibers scattered throughout the absorbent material. When applying the sample to the absorbent surface, the fibers are parallel to each other and to the direction of sample flow. Preferably, the fibers are located adjacent to the pores of the absorbent. Therefore, the fibers tend to direct the sample stream into the capture or binder area rather than radiating the sample stream radially from the capture area. The above embodiments are particularly useful when the absorbent is divided into multiple test areas because the fibers help prevent cross-flow of the sample.

When using a porous plastic or ceramic absorbent, the fibers are preferably silica fibers coated with a ceramic material. When using compressed fiber discs as described above, the discs have cellulosic fibers parallel to each other and to the direction of sample flow.

According to the present invention, the assay can be carried out by placing a binding agent (antigen, hapten or antibody) for the analyte on at least part of the surface of the absorbent. The binder can be placed on the absorbent surface in the form of spots to create the symbolic form or applied on the membrane surface, sprayed or printed. The binder can be placed directly on the support surface (either by passive or covalent methods) or it can be supported on solid particles, such as latex particles, placed on the absorbent surface. Then
The absorbent surface is contacted with the sample suspected of containing the analyte. The binder supported on the surface of the absorbent material is analyte-specific. The analyte present is bound to the binder on the surface of the absorbent material and forms an analyte-binder complex. The surface of the absorbent is also contacted with the tracer simultaneously with or subsequent to contact with the sample. The tracer is a labeled form of the ligand and the ligand used is determined depending on the assay format. In competitive assays, the tracer ligand is the ligand that binds the binding agent for the analyte. In the sandwich assay, the tracer ligand binds to the analyte (direct assay) or the binding agent for the analyte (indirect assay). When using a sandwich assay, the tracer is preferably a labeled form of the analyte-specific ligand. In this assay, the analyte and tracer bind to the supported binding agent (tracer binds directly to the supported binding agent in the competitive assay and to the supported binding agent via the analyte in the sandwich assay). Bond) and consequently remain on the surface of the support. Binding occurs while the sample and tracer pass through the supported binder, and the unbound moieties flow into the absorbent by capillary movement by the absorbent. For example, typical flow rates can be from about 12 seconds / ml to about 40 seconds / ml, although the invention should not be limited thereto.

It is also possible to wash the surface bearing the binder after one or more steps and prior to the steps which follow them. For example, in the case of a sandwich assay, the surface can be washed after sample addition and before tracer addition and / or after tracer addition and before its expression. The wash solution can contain standard aqueous buffers; for example, phosphate and Tris buffers. The wash solution may also contain detergents and chaotropic agents to minimize non-specific binding.

The method of measuring the analyte in the sample to be assayed depends on the type of marker used in conjunction with the ligand or tracer. When using a dye as a label or marker, the dye appears on the absorbent surface and remains on the absorbent surface after any washing steps. Enzyme labeling uses a suitable substrate to develop the color. Radioactive,
The presence of fluorescent, chemiluminescent, enzymes, chromogens, spin labels, biotin, gold particles or other types of markers that remain on the surface of the absorbent material can be measured by any method known in the art. .

The assay can be a qualitative or a quantitative assay, and the term "measurement" as used herein means a qualitative and / or quantitative measurement of an analyte.

Representative examples of specific analyte assays include mononucleosis heterophile antibodies and Group A Streptococcus assays. In an example of a mononucleosis heterophilic antibody assay, a specimen suspected of having mononucleosis heterophilic antibodies is applied to a surface of an absorbent material having a binder or antibody and passed through the binder. After flowing, it is absorbed in the support. The binder is a coated particle (ie,
It can be bound (either passively or covalently) to the absorbent surface, directly to the absorbent surface, or directly or passively or covalently to the absorbent material. When a mononucleosis heterophile antibody is present, it is entrapped in the binder (purified mononucleosis antigen). A tracer that is anti-human IgM or anti-human IgG is then applied to the surface and bound to the human IgM or IgG heterophile antibody bound to the binder. The tracer is, for example, a radioisotope of iodine, cobalt or tritium, an enzyme, a fluorescent dye, an absorbing dye, a metal, a chemiluminescent substance, a spin label, biotin, a gold particle, or a person having ordinary skill in the art. It can be labeled with a variety of detectable markers such as any other known labeling substance. If the heterophilic antibody is not present in the sample, the tracer will not bind to the absorbent surface at a detectable value. The unbound tracer can be removed using a wash step. The washing step may require the use of a combination of detergent and chaotropic agent to minimize non-specific binding of the absorbent moieties to the surface. Wash steps can also be omitted from the assay according to the signal to noise ratio. For example, dye-based systems may use the conjugate in a diluted form in sufficient quantity to enhance the ratio. In enzyme-based systems, the conjugate and / or substrate can double as a wash mechanism. With the tracer made of dye conjugate, the reaction can be visualized immediately after the wash buffer is absorbed into the device. When the enzyme is used as a tracer, the addition of the enzyme substrate results in a color that is visualized on the absorbent device surface. In the absence of mononucleosis antibody, the labeled antibody will not bind to the binding agent. In this case the enzyme does not bind to the surface and does not produce a color.

In the example of an assay for Group A Streptococcus, a sample swab suspected of containing Group A Streptococcus was placed in an extraction buffer (ie, chemical or enzymatic) known to one of ordinary skill in the art. Suspended in. After a single hour of incubation, the Group A-specific carbohydrate antigen is exposed and the extract can be neutralized to improve antigen-antibody binding. Part of the extract is applied to the surface of the absorbent material with the binder. The binder can be bound to the coated particles (ie, latex or other suitable microspheres) on the absorbent surface or directly to the absorbent material by passive or covalent methods. The binding agent can be a purified polyclonal antibody prepared against a Group A specific carbohydrate antigen. Once the extract has passed through the binder, the Group A antigen, if present, is captured by the antibody-containing binder. A tracer, which can be a polyclonal or monoclonal antibody specific for the Group A carbohydrate, is then applied onto the absorbent material and absorbed into the device. In this case, the tracer can be labeled with a variety of detectable markers such as those described above or any other labeling substance known to one of ordinary skill in the art. If the Group A antigen is not present in the extract, the tracer will not bind to the absorbent surface at any detectable value. Unbound tracer can be removed using the washing steps described above. The washing step may need to be used in combination with detergents and chaotropic agents to minimize non-specific binding of the absorbent moieties to the surface. With the tracer made of dye conjugate, the reaction can be visualized immediately after the wash buffer is absorbed into the device. When using the enzyme as a tracer, the addition of an enzyme substrate produces a color that binds to the surface of the absorbent device. In the absence of Group A Streptococcus antigen, the labeled antibody will not bind to the binder. In this case, the enzyme does not bind to the surface and produce no color.

The following example illustrates an embodiment relating to the formation of a porous plastic support according to the present invention and an assay using this support.

[0045]

EXAMPLES Porous plastic supports are made from a mixture of liquid polyethylene and a surfactant. The mixture of liquid polyethylene and surfactant is cast into a cylindrical plastic support having a pore size of about 5 microns to about 30 microns. The slurry of acetyl cellulose is then added to the surface of the plastic support. Acetylcellulose is absorbed in a plastic support and dried. Acetylcellulose is about 0.1 to about 10
Add in an amount to bring pore sizes up to micron on the support.

The hydrophobic plastic support is then contacted with a Verion surfactant. Surfactants are added to the support surface, thereby saturating the support to provide a hydrophilic surface.

The solid support is prepared for use in connection with the mononucleosis assay as follows:

Polystyrene latex particles are covalently coated with mononucleosis antigen prepared from bovine cells. The mononucleosis antigen used in this example was purchased from Meridian Diagnostics. The latex particles are then placed on a portion of the solid support surface. Polystyrene latex particles passively coated or loaded with human IgM are then placed on another part of the solid support. This part is inserted into the part of the support on which the polystyrene latex particles coated with the mononucleosis antigen are placed. The area where the latex particles coated with the mononucleosis antigen and the polystyrene latex particles coated with human IgM are located is in the form of a "+" sign.

The surface of the porous plastic support is then contacted with 0.5% goat serum to prevent non-specific absorption.

The assay is then carried out as follows: 150 μl of human serum is placed on the surface of the porous plastic support and absorbed into the support. Then
100 μl colloidal gold conjugated to goat anti-human IgM
On a porous plastic support. Then 250-500 μl of 1 M guanidine and 5% Triton-X wash buffer are added. The surface of the porous plastic support is then read to determine the presence of mononucleosis antibody. When a mononucleosis antibody is present in the sample, the conjugate of colloidal gold and goat anti-human IgM binds the mononucleosis antibody that binds the mononucleosis antigen as well as human IgM. A plus "+" sign appears on the surface of the support. If the mononucleosis antibody is not present in the sample, the conjugate will bind only to human IgM and a minus "-" sign will appear on the support surface.

In some cases, it is possible to use the porous material in the assay without the analyte binding agent. In such cases, the analyte is retained on the surface of the porous absorbent material, eg, porous plastic. Tracers are also applied to the porous plastic and bind to the analyte if present. The presence and / or amount of analyte is then measured by the presence of tracer on the surface.

However, it should be understood that the scope of the assay of the present invention is not limited to the particular embodiments described above. The present invention may be practiced other than as specifically described and is still within the scope of the following claims.

[Brief description of drawings]

1 is a top view of an embodiment of an absorbent material surface according to an embodiment of the present invention. FIG.

FIG. 2 is an exploded view of the first embodiment of absorbent material contained within a container.

FIG. 3 shows another test area according to the invention.
FIG. 7 is an isometric view of one embodiment.

FIG. 4 is a top view of another embodiment of an absorbent material surface according to an embodiment of the present invention.

FIG. 5 is an isometric view of yet another embodiment representing an absorbent test plate having multiple test areas in accordance with the present invention.

6 is a cross-sectional view of the embodiment shown in FIG.

FIG. 7 is a top view of a plurality of absorbent test plates contained in a tray.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Richard P. McPartland New Jersey 08502, Bellmead, Walnut Grove Road 7

Claims (35)

[Claims] 1. An analyte-containing sample is applied to the surface of an absorbent material having at least one binder for the analyte, supported on at least a portion of the surface of the absorbent material, the absorbent material being on the surface thereof. At least 0.1 micron and 10
Uncharged particles having a size not exceeding micron can be retained, whereby the sample flows through the supported binder to the absorbent material and the analyte binds with said binder; An analyte measurement assay comprising measuring an analyte bound to a binding agent. 2. The assay of claim 1, wherein the assay is a competitive assay. 3. The assay of claim 1, wherein the assay is a sandwich assay. 4. The assay according to claim 1, wherein the assay is the ELISA method.
Assay as described in. 5. The assay of claim 1, wherein the assay is an agglutination assay. 6. The assay of claim 1, wherein the absorbent material is selected from the group consisting of polyolefins, polyesters and acetyl cellulose. 7. The assay of claim 6, wherein the absorbent material is polyethylene. 8. The assay of claim 1, wherein the surface of the absorbent material is treated with a cellulose-containing material. 9. The assay of claim 1, wherein the absorbent material is a compressed fibrous material. 10. The assay of claim 1, wherein the absorbent material is a porous ceramic material. 11. A container comprising an upper lid portion having an opening and a bottom portion, and an absorbent material contained within the container, the absorbent material being at least 0.1 micron.
A device having a surface capable of holding uncharged particles having a size not exceeding 0 micron, the surface being surrounded by an opening in the upper lid portion. 12. The device of claim 11, wherein the absorbent material is selected from the group consisting of polyolefins, polyesters and acetyl cellulose. 13. The device of claim 12, wherein the absorbent material is polyethylene. 14. The device of claim 11, wherein the surface of the absorbent material is treated with a cellulose-containing material. 15. The device of claim 11, wherein the absorbent material is a porous ceramic material that is generally microporous and non-macroporous. 16. The device is used for assaying an analyte and the surface of the absorbent material has at least one binding agent for the analyte supported on at least a portion of the surface. The device according to claim 11. 17. In an analyte assay, an analyte-containing sample is applied to the surface of a wettable porous plastic, the surface being uncharged particles having a size of at least 0.1 micron and no greater than 10 microns. And measuring the analyte retained on the surface. 18. An absorbent material partitioned into a plurality of test areas, each of the plurality of test areas being at least 0.
A device having a surface capable of holding uncharged particles having a size of not more than 10 microns at 1 micron. 19. The device of claim 18, wherein the absorbent material is selected from the group consisting of polyolefins, polyesters and acetyl cellulose. 20. The device of claim 19, wherein the absorbent material is polyethylene. 21. The device of claim 18, wherein the surface of the absorbent material is treated with a cellulose-containing material. 22. The device of claim 18, wherein the absorbent material is a porous ceramic material that is generally microporous and non-macroporous. 23. The device of claim 18, further comprising a cover having a plurality of openings each located on top of a corresponding test area. 24. The device of claim 18, comprising a plurality of grooves separating the test areas. 25. The device is used for assaying an analyte and each surface of the plurality of test areas has at least one binding agent for the analyte supported on at least a portion of the surface. The device according to claim 18. 26. An absorbent material having a surface capable of holding uncharged particles having a size of at least 0.1 micron and not greater than 10 microns, the surface of the absorbent material having at least one well portion. And the at least one well portion is capable of receiving a sample and each of the at least one well portion defines a test area. 27. The device of claim 26, wherein the surface of the absorbent material has a plurality of well portions. 28. The surface of the absorbent material is at least 1.
28. The device of claim 27, having three grooves, each of the at least one groove being disposed between two of the plurality of wells. 29. The device of claim 26, wherein the absorbent material is selected from the group consisting of polyolefins, polyesters and acetyl cellulose. 30. The device of claim 29, wherein the absorbent material is polyethylene. 31. The device of claim 26, wherein the surface of the absorbent material is treated with a cellulose-containing material. 32. The device of claim 26, wherein the absorbent material is a porous ceramic material that is generally microporous and non-macroporous. 33. The device of claim 26, wherein the device is used for assaying an analyte and the surface of the at least one well portion has at least one binding agent for the analyte. 34. The entire material is such that it is entirely microporous, has no macropores, and is capable of retaining uncharged particles having a size of at least 0.1 micron and no greater than 10 microns. A device consisting of a porous ceramic absorbent material having a uniform porosity throughout. 35. The device of claim 34, wherein the porous ceramic absorbent material has pores sized from about 0.5 to about 10 microns.