WO1990015328A1

WO1990015328A1 - Improved immunoassay

Info

Publication number: WO1990015328A1
Application number: PCT/US1990/003222
Authority: WO
Inventors: Martin Gould; Sudhakar Vulimiri
Original assignee: Ampcor, Inc.
Priority date: 1989-06-06
Filing date: 1990-06-06
Publication date: 1990-12-13
Also published as: AU5825790A

Abstract

The present invention comprises a solid phase component of an immunoassay that utilizes the carrier member to separate an immunologic complex from a reaction solution, the carrier member comprising an insoluble solid material to which an effective amount of an immunologically active agent has been bound. The immunologically active agent is bound to the carrier member at a concentration of at least approximately 0.03 νg of immunologically active agent per mm2. In addition, the solid phase component of the immunoassay can be manufactured by a process comprising the steps of bonding the immunologically active agent to a substrate, treating the substrate with an effective amount of serum, treating the substrate with an effective amount of a solution of a protein, and then treating the substrate with a buffer solution. The solid phase components can be utilized in any type of immunoassay which utilizes a solid phase to separate labeled species from the assay medium. The immunoassay is suitable for readily and accurately detecting or quantitating the presence of an analyte substance in liquids or fluids such as body fluids, culture media, food, water and the like without the need for multiple steps or washing.

Description

IMPROVED IMMUNOASSAY

Reference to Related Application This application is a continuation-in-part of U.S.

Patent Application Serial No. 447,594 filed on December 8, 1989, which is a continuation-in-part of U.S. Patent Application Serial No. 361,878 filed June 6, 1989.

Field of the Invention The present invention relates to diagnostic devices and methods and, more particularly, to immunoassay diagnostic devices and methods for readily detecting or monitoring the presence of antigenic substances in fluids such as body fluids, culture media, food, water, and the like. Background of the Invention

The term "body fluids" as used herein includes, but is not limited to, whole blood, blood serum, blood plasma, synovial f uid, urine, cerebral spinal fluid, seminal fluid, tears, pancreatic secretions, bile, liver secretions, saliva, pleural fluid, pericardial fluid, nasal secretions, and feces suspension. The term "analyte" means any substance that will specifically bind to an antibody. The analyte may or may not be the antigen that was used to produce the antibody. It is to be understood that the citation of art contained herein is in no way to be construed as an admission that the art is suitable reference against the present patent application, nor should this citation act as a waiver of any rights to overcome the art which may be available to the applicant

Immunoassay procedures have replaced many other procedures used for in vitro diagnostic methods to detect or quantitate a variety of analytes or antibodies in fluids. The high sensitivity and specificity achieved with immunoassays result from the specific, high-affinity, reversible binding of analytes to antibodies and from the existence of methods for attachment of sensitive detectable labels (radioactive isotopes, fluorophores, ferritin, free radicals, bacteriophages and enzymes) to antibodies or analytes. Radioactive isotopes and enzymes are currently the most extensively used labels, although the use of enzymes is generally preferred. When an enzyme is used as a label in an immunoassay utilizing a solid phase or solid carrier member, the assay is often referred to as an enzyme linked immunometric assay or "ELISA". The number of sensitive, specific immunoassays employing enzyme "tags" is expanding rapidly.

Immunoassay techniques are based upon the binding of the analyte being assayed with an antibody or antibodies forming a complex. One or the other member of the complex may be labeled, permitting the detection or quantitative analysis of the target substance by virtue of the label activity.

Immunoassays are generally classified into two groups: 1) the heterogeneous immunoassay, in which a labeled analyte or antibody is separated from the labeled analyte-antibody complex before measurement of label activity in either fraction; and 2) the homogeneous immunoassay, in which the activity of labeled analyte is measured in the presence of labeled analyte-antibody complex. Two such diagnostic assay techniques used to determine the presence or amount of an analyte in body fluids are generally known as "competitive" assays and "non-competitive" or "sandwich" assays. Typically, in "competitive" assay techniques, an unlabeled antibody preparation bound to a solid support or carrier is first reacted with a labeled analyte reagent solution. The body fluid with the unknown concentration of analyte therein is then added, and the analyte in the sample competes with the labeled analyte for sites on the supported antibody. The amount of labeled analyte reagent displaced indicates the quantity of analyte present in the fluid sample being assayed.

In the case of the "sandwich" or "non-competitive" assay, a quantity of unlabeled polyclonal or monoclonal antibody is bound to a solid support or carrier surface. The bound antibody is reacted with a body fluid sample being evaluated for analytes. After incubation and washing, the sample is incubated with a solution of labeled antibody. The labeled antibody bound to the solid carrier member in an antibody-analyte-antibody sandwich or the amount of unbound labeled antibody in the liquid phase would be determined as a measure of the presence of analyte in the test sample.

The availability of monoclonal antibodies that are specific for a particular analyte has made possible the modification of immunoassay procedures such as disclosed, for example, in U.S. Patents 4,376,1 iϋ and 4,486,530 to David, et al., wherein antibodies specific to analytes could be employed to detect such analyte in body fluids, reducing or eliminating certain intermediate steps in the assay.

Over the years, many attempts have been made to develop more accurate, sensitive, and definitive tests and devices for immunoassay diagnostic testing. For example, in U.S Patent 4,168,146 to Grubb et al., U.S. Patent 4,200,690 to Root et al., and U.S. Patent 4,373,932 to Gribnau et al., immunoassays are described for the detection of the presence of various analytes in body fluids and the like. These immunoassays employ reagents such as antibodies bound to a variety of porous support or carrier materials and dye labeled antibodies or the like. Such devices and methods, however, are generally directed to immunoassays for specific antigenic substances using particular types of porous carrier materials and labels for the antibody reagents which exhibit limited sensitivity and accuracy. Further, such devices and methods require several operations, including washing or quenching steps and extended periods for incubation, which limit their suitability for use by less skilled personnel in a doctors office or in a home care situation.

More recently, procedures for conducting particular types of immunoassays in a relatively short period of time and in a routine fashion which employ generally simple apparatus have been disclosed, for example, in U.S. Patents 4,632,901 and

4,727,019 to Valkirs et al.', U.S. Patent 4,639,419 to Olson et al.;

U.S. Patent 4,703,017 to Campbell et al.; and U.S. Patent 4,786,589 to Rounds. However, while such procedures and devices reduce the complexity and time for certain assays, several steps including a washing or quenching step are still generally necessary, the sensitivities thereof are limited, and the test specimens are generally not suitable for extended periods of storage. Thus, even farther simplification or efficacy is desirable.

One of the persistent problems encountered in immunoassays which employ a solid carrier member is the non¬ specific binding of labeled components in the system to the solid support. Several U.S. Patents address the issue of blocking non¬ specific binding of the labeled components in an immunoassay. Examples of these patents include U.S. Patent No. 4,200,690 to Root et al., which discloses a protein-coated membrane filter for attaching antibodies to the membrane. In particular, the patent discloses coating a membrane filter with the protein zein and then immersing the protein in the desired antibody mixture to bind those antibodies to the membrane filter. In addition, the Root patent discloses including fetal calf serum in the conjugate solution to block non-immunochemical binding of labeled components in the assay to the coated surface.

U.S. Patent No. 4,786,589 to Rounds discloses the need for a blocking agent and discloses the use of a dilute solution of serum albumin as such a blocking agent to reduce the problem of non-specific binding of labeled species.

U.S. Patent No. 3,888,629 to Bagshawe discusses the problem of non-specific binding of the labeled species and uses a dilute bovine albumin solution in the assay to block the non¬ specific binding.

U.S. Patent No. 4,496,654 to Katz et al. also discusses the problem of non-specific binding of labeled species in immunoassays. Katz et al. tried to reduce non-specific binding of the labeled compounds by blocking the solid supports after the reaction with 50 mM ethanolamine, pH = 8.0, at room temperature for 2 hours.

U.S. Patent No. 4,612,281 to Desmonts et al. discloses an immunoassay for detecting disease associated immunoglobulin, such as IgM, associated with acute forms of Toxoplasma gondii infection. The immunoassay utilizes microtiter plates which are blocked with a 5% solution of calf serum in phosphate buffered saline. The patent teaches that the plate surface can be washed with a protein solution such as albumin, bovine serum, or similar proteins.

However, none of these methods of blocking the solid carrier member in an immunoassay are successful in blocking non-specific binding of the labeled components in an immunoassay. Because of this non-specific binding, there is associated with the solid carrier member a high background signal requiring the incubations to be unacceptably long and also requiring extensive washing of the solid carrier member to try to remove the components that are non-specifically binding to the solid carrier member.

Thus, the processes heretofore typically employed require measured quantities of reagents and controlled extended reaction times, as well as several washings and quenching, limiting these procedures to hospitals and laboratories where trained personnel and suitable equipment are available to perform the assays. The desirability of a relatively simple procedure and apparatus which make such assays readily possible in the physicians office or even for use by lay persons in home health care programs is therefore evident.

What is needed is a method of eliminating the non-specific binding of components in an assay system. If the nonspecific binding could be eliminated or nearly eliminated, then the background color or signal would be minimal, thereby allowing detection of a signal from the labeled component of the assay at a much earlier time in the assay. This would make the assay time shorter and would also increase sensitivity of the assay.

Summary of the Invention

As will be seen, the present invention overcomes these and other problems associated with prior art immunoassays. Stated generally, the present invention comprises an immunoassay which is fast, simple, has no washing steps, and maintains the specificity and sensitivity of immunoassays. The immunoassay is suitable for readily and accurately detecting or quantitating the presence of an analyte substance in liquids or fluids such as body fluids, culture media, food, water and the like without the need for multiple steps or washing.

In one aspect, the present invention comprises an immunoassay process which employs a test strip or the like for detecting or monitoring the presence of antigenic substances in fluids such as body fluids and the like, which test strip can be employed by immersion in an assay. The test strip can be employed to visually detect or quantitate the presence of antigenic substances in fluids such as body fluids and the like by immersion in the assay and can be retained as a record of such assay results. Desired results can be obtained quickly and without the need for washing, quenching, multiple steps, or extended delays. The process can be readily carried out in a physician's off "e or in home health care situations, as well as in hospitals am. clinical laboratories.

Stated somewhat more specifically, the present invention comprises a method of improving both competitive and non-competitive immunoassays that utilize a solid phase component for separating labeled complexes from other components in an assay. The invention eliminates the non-specific binding of components in an assay system, thereby minimizing the background noise and permitting the detection of the signal from the labeled component in the assay in a much shorter time and with greater sensitivity.

In one aspect, the present invention comprises a process for treating a solid phase component utilized in immunoassays which provides the solid phase component with significantly improved blocking characteristics over prior art immunoassays. In another aspect, the invention comprises a solid phase component which exhibits the improved blocking characteristics. In yet another aspect, the present invention comprises an immunoassay process for detecting with significantly enhanced speed and sensitivity whether a target immunologically active agent is present in a fluid sample.

The solid phase component of the immunoassay system of the present invention comprises an improved blocking arrangement for minimizing the non-specific binding of components in an assay system. In one aspect of the invention, a process is disclosed for preparing the solid phase component for use in an immunoassay that utilizes the solid phase component to separate an immunologic complex from a reaction solution. An effective concentration of an immunologically active agent is first bound to the surface of a carrier member or other substrate. In the disclosed embodiment, the effective concentration is at least 0.03 μg of the immunologically active agent per mm². In one embodiment, the immunologically active agent comprises an antibody; in another embodiment, the immunologically active agent comprises an analyte.

To block the bound immunologically active agent against non-specific binding of components in the assay system, the carrier member of the disclosed embodiment is treated by an improved process. First, the carrier member with bound immunologically active agent is treated with an effective amount of a serum such as goat serum. Next, the carrier member with bound immunologically active agent is treated with an effective amount of a solution of a protein, such as casein. Optionally, the serum and the protein can be combined in one solution. The carrier member with bound immunologically active agent is then treated with a buffer solution, preferably a borate buffer. The buffer solution may optionally contain a sugar, such as mannitol, dissolved therein. In certain immunoassays, the carrier member with the immunologically active agent bound thereto may be blocked with serum only. Furthermore the serum does not intefere with the assay procedure. When so treated, only components in the assay system which are specific to the bound immunologically active agent bind to the carrier member.

To conduct an assay according to the present invention, a labeled capture reagent against a target immunologically active agent to be assayed is introduced into a fluid sample to be assayed. The fluid sample and the labeled capture reagent are then brought into contact with the carrier member with the capture reagent bound thereto. The quantity of the labeled capture reagent which binds to the carrier member provides an indication of the quantity of the target immunologically active agent in the fluid sample. Because the treated carrier member effectively blocks non-specific binding of components in the assay system, the background noise is significantly reduced, and the signal from the labeled component in the assay can be detected in a much shorter time and with greater sensitivity than prior art immunoassays. Because immunoassay procedures vary widely, the blocking procedure described herein may be varied to maximize sensitivity of the assay.

Immunoassays in which the present invention can be used include assays wherein the solid carrier member portion of the immunoassay is treated so that non-specific binding of labeled analyte or antibody is inhibited thereby allowing early detection of a positive reaction. These assays include, but are not limited to, agglutination immunoassays, immunosorbent immunoassays and dipstick immunoassays. By employing the blocking step in a conventional immunoassay, the time and number of steps required to complete the assay is reduced significantly.

Accordingly, it is an object of the present invention to provide an immunoassay that is fast, simple, does not require a wash step or a quench step, and maintains the specificity and sensitivity of immunoassays.

It is another object of the present invention to provide an immunoassay that can be used to detect analytes in urine.

It is another object of the present invention to provide a method of treating the solid carrier member in an immunoassay that will limit or eliminate non-specific binding of components in the assay. It is yet another object of the present invention to provide an immunoassay which is suitable for readily and accurately detecting or monitoring the presence of an analyte substance in liquids or fluids such as body fluids, culture media, food, water and the like without the need for multiple steps or washing.

It is yet another object of the present invention to provide an immunoassay which is suitable for readily and accurately detecting or monitoring the presence of an antibody substance in liquids or fluids such as body fluids without the need for multiple steps or washing.

It is another object of the present invention to provide an immunoassay kit which employs a test strip or the like for detecting or monitoring the presence of antigenic substances in fluids such as body fluids and the like, which test strip can be employed by immersion in an assay and desired results can be obtained without the need for washing, quenching, multiple steps or extended delays.

It is a further object of the present invention to provide a test device such as a "dip-stick" type test strip which can be employed to visually detect or monitor the presence of antigenic substances in fluids such as body fluids and the like by immersion in an assay and can be retained as a record of such assay results. It is a still further object of the present invention to provide a process for the immunoassay of fluids such as body fluids and the like for the detection or monitoring of antigenic substances which can be readily carried out in a physicians office or in home health care situations as well as in hospitals and clinical laboratories. It is another object of the present invention to provide a process for the immunoassay of fluids such as body fluids and the like for the detection or monitoring of an analyte which can be readily carried out in one or two steps with readily available equipment and wherein the results can be accurately determined visually by a test strip or similar device.

These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

Brief Description of the Drawings

For the purpose of illustrating the invention, there is shown in the accompanying "wing one form which is presently preferred; it being understood

the invention is not intended to be limited to the precise arrangements and instrumentalities shown.

Figure 1 is a perspective view of an immunoassay diagnostic kit in accordance with the invention.

Figure 2 is a perspective view of a "dip stick" test device in accordance with the invention.

Figure 2a is an enlarged perspective view, partly broken away, of the "dip stick" test device of Figure 2.

Detailed Description of the Invention

The present invention comprises an immunoassay process which employs a test strip or the like for detecting or monitoring the presence of analytes in fluids such as body fluids and the like, which test strip can be employed by immersion in an assay. The test strip can be employed to visually detect or quantitate the presence of antigenic substances in fluids such as body fluids and the like by immersion in the fluid and can be retained as a record of such assay results. Results can be obtained quickly and with the need for washing, quenching, multiple steps or extended delays. The process can be readily carried out in a physician's office or in home health care situations, as well as in hospitals and clinical laboratories. In the following specification and claims the term

"analyte designates all substances that can react specifically with an antibody such as proteins, polysaccharides, and hormones, and antigens associated with bacteria, surface components of bacteria, fungi, ricketsia, protozoa, viruses. Analytes also designates lower molecular weight chemicals such as alkaloids, steroids, barbiturates, aminoalkylbenzenes, purines, vitamins, antibiotics, aminoglycosides, and peptide hormones. The present invention is especially useful for detecting antigens in urine that are associated with microorganisms that cause urinary tract infections. These microorganisms include, but are not limited to,

Enterobacter sp., Enterococcus, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Proteus sp., Staphylococcus epidermidis (albus), cc-hemolytic streptococcus, and β-hemolytic streptococcus.

Antibodies are proteins which exhibit a specific immunological activity against the antigen which caused their formation. Antibodies are very similar in their overall protein structure, but are distinguished from each other by their specific affinity to different analytes. In the following specification and claims the term "antibody" is meant to denote a proteinaceous material which exhibits antibody activity, that is, the antibody in substantially pure form or in form of a mixture having a high antibody-activity, e.g., an anti-serum.

In the following specification and claims the term "immunologically active agent" is meant to include analytes, antibodies, and immunologically active binders.

Immunometric assays have been found to be particularly well suited for the detection of polyvalent analytes, i.e., substances that are able to complex with two or more antibodies at the same time. Such assays employ a quantity of unlabeled antibody bound to a solid support that is insoluble in the fluid being tested and a quantity of soluble antibody bearing a label, such as a radioactive isotope or an enzyme, that permits detection or a quantitative estimate of the amount of the ternary complex formed between the solid carrier member antibody, analyte, and labeled antibody.

Immunometric assays known in the prior art typically employ a "forward" assay, in which the antibody bound to the solid carrier member is first contacted with the sample being tested to extract the analyte from the sample by formation of a binary solid carrier member antibody analyte complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted analyte if any, and then contacted with a solution containing a known quantity of labeled antibody.

After a second incubation period to permit the labeled antibody to complex with the analyte bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. In a simple qualitative to determine whether the analyte is present in the sample being tested, the washed solid support is tested to detect the presence of labeled antibody, for example by measuring the emitted radiation if the label is a radioactive element. The amount of labeled antibody detected is compared to that for a negative control sample known to be free of analyte. Detection of labeled antibody in amounts substantially above the background levels indicated by the negative control indicates the presence of the analyte. Quantitative determinations can be made by comparing the measure of labeled antibody with that obtained for standard samples containing known quantities of the analyte. In addition to the "forward" immunoassay, many variations of the immunoassay have been developed over the past several years. The simultaneous assay involves a single incubation step as the antibody bound to the solid support and the labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

A reverse assay involves the stepwise addition first of a solution of labeled antibody to the fluid sample, followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period. After a second incubation, the solid carrier member is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with the solid support is then determined as in the simultaneous and forward assays.

Both the simultaneous and reverse assay techniques require a sufficient excess amount of solid carrier member antibody to bind most or all of the analyte present to avoid an artificially negative or low quantitation of analyte.

These immunoassays were first performed using polyclonal antibodies. Large amounts of highly purified, active antibody for preparing a solid carrier member with sufficient analyte binding capacity is difficult to obtain from the "polyclonal" antibodies used in prior art processes. This problem was addressed in the U.S. Patent Nos. 4,376,110 and 4,486,530 to David, et al.. These patents disclose increasing the specificity and speed of the assay by using monoclonal antibodies as both the antibody on the support and the labeled antibody, rather than polyclonal antibodies. None of the these prior art immunoassays solve the problem of non-specific binding of the labeled components to the solid carrier member. Many attempts have been made to reduce the non-specific binding of the labeled components. These include incubation of the solid carrier member in serum or in protein solutions, usually a solution of bovine serum protein. Even after treating the solid carrier member with the prior art methods, the non-specific binding of labeled analyte increases the time the assay must be performed and decreases the sensitivity of the assay.

The present invention addresses the problem of non¬ specific binding of the labeled components in the assay medium by treating the solid carrier member in an immunoassay in such a way that non-specific binding of the labeled component is reduced to a minimum. According to the present invention, the solid carrier member is treated in a multistep process. After treatment, the solid carrier member can be stored for long periods of time at room temperature or other temperatures without reducing the efficacy of the assay.

In a preferred embodiment of the present invention, the solid carrier member of an immunoassay is treated first with a solution of serum. In the disclosed embodiment, the serum is preferably goat serum. However, it is to be understood that the source of the serum includes, but is not limited to, goat, rabbit, bovine, pig, horse, calf, mouse serum, fetal calf or human serum.

The solid carrier member is preferably contacted with the serum for several minutes several hours. The concentration of the serum should be between approximately 1% and 100% and more preferably between approximately 50% and 100% serum.

After contacting the solid carrier member in serum, the solid carrier member is then contacted with a solution of a protein such as casein. Preferably, the casein is Hammarsten casein (BDH Chemicals Ltd., Poole, England). (See Hammarsten, et al., Z. Physio. Chem. Vol. 7, page 227, (1883) and Hipp, et al., /. Dairy Science, Vol. 35, p. 272 (1952)) The concentration of casein is preferably between about 0.1% to 25% with the most preferable concentration of approximately 0.3% casein. The solution of casein should be before use to remove any particles that may be in the solution. All of the reagents used in the immunoassay must not be contaminated with any of the analytes that are to be tested. The solid carrier member should remain in contact with the casein solution for approximately 1 to 10 minutes. Optionally, the solid carrier member can then be dried at room temperature for several minutes to several hours. The solid carrier member can be dried at temperatures other than room temperature. It is to be understood that it is contemplated as part of the present invention that the serum and the casein can be combined in a single solution, and the solid carrier member can be immersed in the single solution to effectively block the non-specific binding of the labeled components of the assay.

Finally, the solid carrier member is washed in a buffer solution such as a borate buffer. Other buffers that can be used include, but are not limited to, tris buffer, phosphate buffer, hepes buffer, and pipes buffer. Optionally, a sugar is dissolved in the wash buffer. The preferable concentration of sugar is between 0.1% and 50% with the more preferable concentration of sugar of approximately 0.5 to 10% and the most preferable concentration of approximately 1%. It is important to note that many different sugars can be used in practicing the present invention. The sugar in the solution is preferably mannitol, but sucrose, sorbitol, corn syrup, glucopyranosides, maltose, fructose, glucose, xylose, and oligosaccharides may be used.

It is to be understood that, for some assays, the solid carrier member with the antibody or analyte bound thereto, can be treated with serum only. It is contemplated as part of the present invention that the treatment protocol for blocking the solid member can vary widely depending upon such factors as the material comprising the solid carrier member, the assay being performed, the labeled capture reagent, as well as many other factors.

An important aspect of the present invention is in the concentration of the immunologically active agent on the solid member carrier. It is important that a high concentration of immunologically active agent be applied to the solid carrier support. This can be done by multiple applications of the antibody solution to the support. A preferred method of applying the immunologically active agent solution is with a thin layer chromatography spotter which applies the solution to the support by multiple passes over the support. One such spotter that can be used to apply the antibody is a Camag TLC spotter device (Camag Scientific Inc., Wilmington, NC). According to the present invention, the concentration of immunologically active agent is at least 0.05 μg/mm². The preferred concentration is 0.3 μg/mm² The immunologically active agent can be an antibody or an analyte. Optionally, the concentration of antibody applied to the solid carrier support can be increased by raising the salt concentration of the antibody solution, thereby increasing the binding activity of the antibody. The salt concentration can be between approximately 0.1 M to 2 M with a preferred concentration of approximately 0.5M. The preferred salt is sodium chloride, but other salts that can be used include, but are not limited to, sodium phosphate calcium chloride, lithium chloride, magnesium chloride and zinc chloride.

In one embodiment of the present invention, the immunoassay comprises contacting a fluid test sample containing a target analyte with a labeled capture reagent that is specific for the target analyte. The labeled capture reagent is usually an antibody that is specific for the target analyte. The labeled capture reagent is preferably labeled with an enzyme, although the reagent can be labeled with any compound that is capable of eliciting a measurable signal. The fluid test sample is also in contact with a capture reagent which is bound to a solid carrier member. The bound capture reagent is usually an antibody that is specific for the analyte.

During an incubation period, the target analyte binds with the labeled capture reagent and the bound capture reagent.

The labeled capture reagent/analyte/bound capture reagent complex is now immobilized on the solid carrier member. The amount of labeled capture reagent bound to the solid carrier member is measured by measuring the signal emitted by the label on the labeled capture reagent. This signal is directly proportional to the amount of target analyte in the fluid test sample.

In another embodiment of the present invention, the fluid test sapiple is admixed with a solution of labeled capture reagent (labeled antibody) that is specific for the target analyte.

After an incubation period, the reaction admixture is brought into contact with the bound capture reagent. The bound capture reagent specifically binds to the analyte/labeled antibody conjugate. The labeled capture reagent/analyte/bound capture reagent complex is now immobilized on the solid carrier member and can b τemoved from the fluid test sample. The amount of labeled capture reagent bound with the solid carrier member is measured by measuring the signal emitted by the label on the labeled capture reagent. This signal is directly proportional to the amount of target analyte in the fluid test sample.

In a more specific example, an analyte present in the fluid test sample will bind to the labeled antibody capture reagent which is specific for the target analyte. When the fluid test sample is contacted with the bound antibody capture reagent, the analyte also binds to available site on the bound antibody to form an antibody-analyte-antibody "sandwich." In accordance with the present invention, evaluation of the signal emitted by the label is an accurate indication of the presence of the target analyte in the sample.

Because the non-specific binding of the labeled components in the fluid test sample to the solid carrier member has been inhibited or eliminated, there is no need for washing or quenching or for extended periods of incubation. In the case of an enzyme labeled antibody capture reagent a solution of color forming substrate which reacts with the enzyme label will render a readily discernible signal. The signal-generating substrate in encapsulated or other time-release form may be included in the body fluid-labeled antibody admixture or may be in a solution separate from the reaction mixture into which the supported complex can be immersed.

In another aspect of the invention, the apparatus of the invention comprises, as a first component, an insoluble solid carrier member to which is bound a desired amount of a bound capture reagent against the target agent being assayed, such as an antibody against a target analyte, generally in a controlled linear, dot-like, or other regular pattern, the carrier member preferably being affixed to the surface of a non-absorbent, inert support. The carrier member is substantially blocked against binding to other immunologically active agents as described hereinabove. The apparatus further comprises as a second component at least one container which is non-absorbent and inert to immunologically active agents in which a fluid test sample and a labeled capture reagent may be admixed and which permits insertion of the first component into the admixture.

The apparatus of the present invention is useful in a variety of immunoassays for readily and accurately detecting or monitoring the presence of antigenic substances in fluids such as body fluids including blood serum, urine, etc.; culture media; food; water, and the like. The apparatus can be provided as a diagnostic kit to permit such assays to be conveniently and accurately performed in a physician's office or in home health care programs, as well as in hospitals and reference and clinical laboratories.

Referring now to the drawings, in which like reference numerals identify like parts throughout the several views, Figures 1, 2 and 2a, illustrate a "diagnostic kit" 10 in accordance with the present invention. The diagnostic kit 10 comprises a "dip-stick" type device or test strip 12. The test strip 12 comprises a thin, insoluble, solid, film-like carrier member

14 extending over and affixed to a portion of a surface of a nonadsorbent, inert support 16 by a suitable adhesive 18 or the like which is inert to immunologically active agents. Bound to a portion of the surface of the thin, carrier member 14 in a linear or any other desired pattern or array is a bound capture reagent

20, preferably a polyclonal or monoclonal antibody preparation or mixture thereof against the target analyte in a fluid test sample. Antibodies suitable for use in accordance with the invention may be prepared by any of the techniques known in the art.

Carrier member 14 can be prepared from any insoluble, solid material to which immunologically active reagent preparations such as antibodies or analytes can be attached by chemical binding or adsorption, and which does not contain functional groups which will interfere with the immunological chemical reaction. Especially suitable are solid non-fibrous materials which will chemically bind with immunologically active capture reag^ts and into which such capture reagents will rapidly diffuse during the coating thereof and when contacted during diagnostic testing. Suitable materials include organic polymers which can used with film forming, blow molding, and other conventional fabrication techniques such as polyethylene, polya ides, e.g., nylon, polypropylene, ethylenepropylene copolymers, polybutylenes and polystyrene. Other suitable materials include halogenated organic polymers such as polyvinyl chloride, polyvinylidene chloride and polytetrafluoroethylene; polyesters such as polyethylene terephthalate, polyacrylates and polymethyl-acrylates; cellulose and cellulose derivatives. Also suitable are fibrous cellulose and cellulose derivatives and non- organic materials such as glass fibers.

Typically, film forming materials are advantageously used which contain carboxyl groups, or primary or secondary amide groups such as polyacrylamide, or which have amino group residues, or into which such groups have been introduced by chemical means, such as nylon, or on which an amide group can be provided, such as polyacrylonitrile having a nitrile group which can be converted to an amide group by known methods.

The thickness and degree of porosity of carrier member 14 are not critical and can vary depending on the particular immunoassay for which the "dip-stick" type test strip or kit is intended. Typically, the solid carrier member is in a thin, preferably non-fibrous, film-like form, generally of 10 mils or less in thickness, having a pore size generally in the range of from about 0.01 micron to about 2 microns, and preferably from about .20 to about .50 microns. However, carrier members of a thickness to about one-quarter inch or having a pore size up to about 100 microns may be employed in certain applications. In addition, the solid, generally film-like carrier member 14 may be attached to the two opposing sides of support 16.

The capture reagent 20 can be bound to the carrier member 14 by any of the well known techniques, such as coupling antibodies, either polyclonal or monoclonal, directly by the well known glutaraldehyde or succinnamide method. Other coupling agents that can be used in the present invention include carboxyldiimidozole, periodate, hydrazide, cyanogen bromide and protein A, protein G, biotin/avidin, and strep/avidin. The amount of capture reagent 20 that is attached to the solid film¬ like carrier member 14 is important but may vary depending on the effective amount required and the affinity thereof to the carrier member which, in turn, is dependent on the immunological reaction in question. Preferably, to enhance the sensitivity of the test and intensity of color formation for detection of the presence of the target analyte, the bound capture reagent 20 will be applied to only a relatively small portion of a surface of the solid carrier member 14 in a controlled linear, dot-like or other desired pattern or array by jet-type atomizer guns and the like which apply a desired quantity of material in a narrow band or array, e.g, a concentration of about 2.5 micrograms of capture reagent per 5 mm of carrier member in a linear band width from about 0.1 mm to about 8 mm, preferably from about 1 mm to about 3 mm, across the full width of the carrier member. In accordance with the practice of the invention, one or more separate bands of capture reagent may be applied and bound to a surface of the solid carrier member 14, thus enabling the detection of one or more target analytes in a sample fluid, preferably by concurrent assays. Bound capture reagents against a variety of different target analytes may be separately bound to the carrier member to facilitate the assays of different target analytes in a fluid test sample with the sensitivity and accuracy of each assay being substantially similar. It would be evident that such concurrent assays, however, would not be suitable in the case where cross-reacting analytes or capture reagents are involved.

In another embodiment, carrier members 14 of the invention may also include a known or control concentration of the target analyte applied to a portion of a surface of the carrier by known techniques in a similar linear, dot-like or the like pattern employed for application of the bound capture reagent 20 to the carrier member but separate therefrom. The presence of such a control reagent on the film-like carrier member can serve as verification that the assay process of the invention was properly carried out, as well as provide a further comparison for detection of the presence of the target analyte in the body fluid test sample.

Support 16 to which carrier member 14 is attached can be any non-absorbent polymeric and the like material which is inert to immunologically active agents and to which the solid film-like carrier member 14 can be conveniently mounted.

The diagnostic kit 10 of the present invention includes a vial 22 or the like container in which an admixture of immunologically active reagents can be reacted and which permits insertion of a dip-stick type device or test strip 12 with the carrier member 14 in contact with the admixture. The vial 22 may be made of glass or a suitable plastic material which is inert to immimologically active agents and non-absorbent. The size of the vial or container 22 is preferably selected so that prescribed amounts of a fluid test sample such as a body fluid and a labeled capture reagent such as a labeled antibody against target analyte in the test fluid may be admixed and reacted, and upon insertion therein of a carrier member 14 having bound antibody, intimate contact thereof with the reagent admixture will be effected. In general, the vial 22 is intended to be disposable after use and to facilitate the disposal of sample and reagents in a simple and hygienic fashion, it is preferred to include a suitable stopper for the vial 22 or envelope therefore (not shown), which can be of any known type. As shown in the figures, additional vials 22 may be included with the kit 10 to facilitate detection of the presence of labeled capture reagent by contact with a color solution separate from the reaction mixture, or to facilitate additional testing cf the fluid test sample. In accordance with the invention, a diagnostic kit 10 may also include stoppered vials 24 and additional dip¬ sticks 12 containing stabilized immunologically active capture reagents to be used in various immunoassays. Such immunoassays, color substrates for detection, standardized test samples for comparison testing, and the like are disclosed, for example, in U.S. Patent No. 4,859,604, the disclosure of which is incorporated herein by reference. As also shown in the figures, the kit 10 may be provided with a base stand 30 and cover 32 to facilitate storage and shipping as well as serving as a stand for the vials 22 when running the assays. As noted hereinabove, in accordance with the practice of the invention the fluid test sample such as a body fluid, culture media, food, water, or the like is admixed with a labeled capture reagent, for example, a monoclonal or polyclonal antibody to a target analyte in the test sample. In the case of an immunoassay, the labeled capture reagent binds to the target analyte in the test fluid during the reaction in the vial 22.

Preferably the labeled reagent is labeled with an enzyme, although other conventional labels may be used in appropriate circumstances, such as, for example, a fluorescent label, a radioactive isotope, latex labels, colloidal conjugates, chemiluminescent labels, bioluminescent labels. Other useful labels may be dyes from which conjugates with capture reagents such as an antibody may be prepared using known techniques.

Useful in the preparation of such conjugates include dyes generally having functional amine groups such as azure A, azure

B, azure C, methylene blue, thionin and the like as described in H.

J. Conn, Biological Stain, 9th Edition, 1977, Williams and Williams Co.

After the fluid test sample and labeled capture reagent have been admixed in the vial 22, the solid film-like carrier memlser 14 having capture reagent 20 bound thereto is immersed in the admixture in vial 22. If, as noted, the fluid test sample contains a target analyte and the labeled capture reagent in the admixture is a labeled antibody to the target analyte, the analyte and labeled antibody bind together. In this case, when the capture reagent bound to the solid film-like carrier member 14 is an antibody against the target analyte, the analyte in the labeled complex will also react with and bind to available sites on the bound antibody with the formation of an antibody-analyte- antibody "sandwich." Depending upon the substrate material used, the presence of the target analyte in the fluid test sample may be signalled by a visual color change after only a brief incubation period (about 1-5 minutes). The color change in the case of labeled antibody reagent occurs by virtue of the use, for example, of a solution of color forming substrate which reacts with the enzyme label as the solution contacts the same in the antibody-analyte-antibody 'sandwich" bound to the carrier member. Such color forming substrate may be present in the admixture in time-release form or may be in a solution in a vial separate from the reaction mixture into which the complex bound to the carrier member can be immersed. In any case, there is no need to wash or treat the carrier member prior to contacting the color forming solution, or to quench the reaction further. The results as evidenced by the "dip-stick" can be retained as a permanent record.

As shown in Figure 2a, the linear pattern depicts the enhanced color development of the bound antibody (capture reagent) compared to the surrounding blocked portions of the carrier member achieved by virtue of the controlled application of the bound capture reagent. This pattern makes readily discernible by visual detection the presence of labeled antibody and target analyte which are bound thereto. The color change results, as noted herein, from the use of any one of a variety of methods for detecting the label on the sandwich complex bound to the carrier member. As would be evident, detection of the presence of a target analyte by instrument measurement and the like of the labeled antibody bound to the solid earner member would be an alternative method for jractice of the present invention. As indicated, similar mea..s may be employed for detecting the presence of more than one analyte in the test fluid by concurrent assays employing appropriate bound and labeled capture reagents against the target analytes. In another embodiment, application of the invention may be carried out by inserting the dip-stick type device 12 with carrier member 14 having bound capture reagent 20 such as an antibody against the target analyte into vial 22 and adding a fluid test sample with a target immunologically active agent (analyte) and labeled antibody capture reagent against the target analyte into the vial 22 for contact with the carrier member 14. While the order of addition of the fluid test sample and labeled capture reagent to the vial or container 22 is not critical, it is generally preferred to add the test sample first

The apparatus of this invention may also be used to perform competitive assays, i.e., assays in which the capture reagent bound to the solid carrier member mounted on the dipstick type device 12 and for which the target immunologically active agent (analyte) in the fluid test sample competes with a fixed quantity of labeled capture reagent added to the fluid test sample or added following sample addition.

The solid support or solid carrier member that can be used to practice the present invention can be comprised of a wide variety of materials. These include, but are not limited to, materials such as nylon, polystyrene, cellulose acetate, glass, polyvinylchloride, microporous silicon/poly vinylchloride, Teflon® and polytetrafluoroethylene, and combinations of these and others are used for the attachment of polyclonal or monoclonal antibodies, enzymes, bacteria, etc.

Attachment of the label to the immunologically active agent, whether that agent be an analyte or an antibody, is a procedure that is well known to those of ordinary skill in the art. Briefly, the procedure is as follows: The enzyme is commonly first dialyzed against a buffer. The antibodies are dissolved in a phosphate buffer. The antibody and the enzyme solutions are combined and allowed to incubate. Glutaraldehyde is added and allowed to react thereby cross-linking the enzyme and antibody. Sodium borohydride is added until degassing subsides. Stabilizers, such as BSA, amino acids, immunoglobulin, and normal serum can then be added. The enzyme conjugate is lyophilized and stored at room temperature.

The covalent attachment methods are also well known to those of ordinary skill in the art. The reagent is usually streaked in a line, but any geometric configuration is contemplated as part of the present invention. The solid support is allowed to dry. A positive and negative control zone can be added to the support using the same attachment protocol. This can be anti-goat or anti-mouse IgG or the actual analyte being tested. The line dimensions can vary in width from 0.01 inches to 0.25 inches or greater, with the preferred width being about 0.06 inches. The length of the line can vary from approximately 0.03 inches to 0.5 inches, with the preferred length being approximately 0.25 inches. The length of the line can vary widely with the only limitation being the sample volumes required to run the test.

The dried solid support is then blocked to preserve the attached antibody and to remove non-specific binding sites.

The solid support is totally immersed in serum. The serum can be from any mammahan species and can vary in concentration of between approximately 0.1% to 100% serum, preferrably 50% to 100%, and most preferably 100%. The buffer that is used to dilute the serum is preferably 0.015 M phosphate buffer at a pH of 7.4. However, many buffers could be used to dilute the serum. The support is immersed in the serum for approximately 3 minutes followed by an immediate submersion in approximately 0.3% filtered Hammarsten casein. Non-fat milk can be used as a substitute for casein. However, casein is preferred. The casein can be used at a concentration of between 0.1 to 25% weight volume. The support is then immersed in a 1% mannitol solution in 0.015 M phosphate buffer at a pH of 7.4. The support is then stored dessicated at a relative humidity of less the 25%. The support can be dried at a temperature between approximately 20° C and 30° C.

It is important to note that the blocking solutions should be filtered prior to use through 0.2 μ nylon and Teflon® filters or equivalent filters. The filtration step is necessary for removing bacteria contamination and also for removing foreign material that clogs the solid support that could interfere in the reactions. It has been found that the filtering step often enhances the speed of the reactions. Thimerosal or sodium azide may be added at a concentration of approximately .02% to prevent microbial growth and to prolong the life of the reagents.

If sample size is of concern, the dipsticks may be saturated with the sample rather than being completely immersed in the sample. If desired, the support may comprise a wick for drawing the sample toward or away from the bound antibody on the support.

The results obtained using the dipstick methodology are both quantitative and qualitative. Semi-quantitative results can be obtained by the comparison of the intensity of the specimen reaction zone to the positive control reference zone.

Additionally, quantitative results can be obtained when the dipstick assay is used in conjunction with a small instrument such as an instrument where the dipstick immunoassay is calibrated against known controls.

The foregoing is a general description of the apparatus and process of the invention, primarily in its use in performing immunoassays which readily provides accurate detection of target analytes in fluid test samples in a short time, e.g., about five to ten minutes. The process can be carried out in one or possibly two simple steps and can be readily performed in home care situations or in hospitals. The invention will now be demonstrated by the description of certain specific examples of its practice. In these examples, unless stated otherwise, all temperatures are centigrade and all parts and percentages are by weight.

Example 1

An antibody specific for the analyte is attached to a nylon solid support. The reagent is streaked in a line across the solid support. It is important that the antibody concentration on the solid support be at least 0.03 μg/mm². Glutaraldehyde is used to bind the antibody onto a nylon film carrier member that has been hydrolysed or treated to promote NH2 groups. The carrier member is treated in 8% glutaraldehyde for a minimum of 0.5 minutes to 12 hours, with 1 hour being preferable. After antibody attachment the use of sodium borohydride or L-lysine to deactivate the unreacted glutaraldehyde is necessary. The carrier members are subsequently washed in distilled water for 2 hours to remove any free glutaraldehyde. They are then allowed to dry at room temperature and are stored dry.

Example 2

Three analytes are assayed using the present invention. These are human chorionic gonadotropin, Salmonella, and E. coli.157. The substrate used is 0.4 g/1 of NBT and 1.6 g/1

BCIP in either 0.1 M AMP Tris or AMPD at pH 9.6 to 9.8. The samples are diluted, used neat, or diluted in nutrient broth, placed in a dried conjugate tube to dissolve dried conjugate for 30 to 60 seconds. The nylon solid support that has bound to it antibodies that are specific for the bacteria, is immersed in goat serum for three to five minutes, followed by immersion in a 0.3% solution of casein (Hamarstein casein from BDH Chemicals Ltd., Poole, England), then immersion in a 1% mannitol solution and then dried. The nylon solid support is then submerged in the sample for 4 to 15 minutes and then immediately placed in substrate for 4 to 5 minutes. A positive result can be seen in about 5 minutes. It is important to note that the support does not need to be washed between the incubation in sample and incubation in the substrate solution. A line develops at the top where the positive control zone is applied. The positive result is a blue-black line at 25 mlU/ml of human chorionic gonadotropin, 200 cells/ml of Salmonella, and 2000 cells/ml of E. coli.157. The remaining portion of the solid support serves as a negative control. The negative control reacted the same as the surrounding membrane and therefore is not apparent.

Example 3

The present invention can be used in agglutination tests. A colored or dyed polystyrene latex particle, charcoal, D- toluidine pigment, or a dyed stroma can be bound/conjugated to antibodies specific for an analyte. In this example, latex particles that are 0.1 microns or smaller are used. The conjugated particle is blocked by incubating the particles in 50% goat serum for 3 minutes, then a solution of 0.3% Hammarsten casein, then a 1% mannitol solution.

Example 4

In this test for detection of the presence of human chorionic gonadotropin (HCG), an analyte which is elevated in urine and blood serum of pregnant women, a dip-stick device with a thin nylon film carrier member is used. The carrier member contains two spaced reaction zones: a positive control zone to which is bound a narrow linear band (1 mm to 3 mm wide) of HCG equivalent to 50 mlU/ml HCG, and a specimen reaction zone to which is bound a narrow linear band (1 mm to 3 mm wide) of a polyclonal antibody sheep) against beta-HCG (affinity purified) at a concentration of 2.5 microgram of antibody per 5 mm of carrier. The bands of reagents are applied to the carrier surface with a thin layer chromatography spotter device. After the reagents dry, the carrier member is immersed in a goat serum for three to five minutes, followed by immersion in a solution of casein (Hammarsten casein from BDH Chemicals Ltd., Poole, England), and then immersion in a 1% mannitol solution. Samples of centrifuged blood serum are admixed with a monoclonal antibody (anti-HCG mouse) to which is bound alkaline phosphatase in sample test vials at room temperature for a short time (about one minute), after which the dip-stick devices are inserted into the reaction admixture in the test vials and permitted to incubate for four to five minutes. The dip-sticks are then placed in a dye solution composed of 0.4 g/1 of nitro blue tetrazolium and 1.6 g/1 of 5-bromo-4-chloro-3-indolyl phosphate for five minutes. The reaction zone linear band placed on the carrier member of the dip-stick will become visible as a distinct blue color if the analyte is present in the sample fluid. The control zone linear band placed on the carrier member will become visible as a distinct blue color of generally similar color intensity to the reaction zone color depending on the analyte concentration in the fluid test sample. No washing or quenching is required to achieve the desired results and the dipsticks can be retained for permanent record purposes.

Example 5

An antibody that is specific for human chorionic gonadotropin is applied to the nylon membrane carrier surface at a concentration of approximately 0.5 μg/mm². The carrier membrane is blocked after 30 minutes. The blocking solution and the timings are as as follows: a) 2 minutes in 0.1 M L-lysine in 0.1 M borate buffer, pH 8.4; b) 30 seconds in 100% goat serum c) 30 seconds in 0.3% Hammarsten casein in 0.015 M KP04, pH 7.4 d) For 20 minutes, the carrier membrane is allowed to dry in less than 25% relative humidity conditions after which the membranes are treated in a 0.1 M borate, pH 8.4 for four minutes. The sample of urine is admixed with the conjugate described in Example 4, and after 1 minute the dipstick is placed in the sample vial and incubated for 2 minutes at room temperature. The dipstick is then placed in the dye solution containing 0.8 g/1 BCIP, 0.3 g/1 NBT in 0.1 M AMP, pH 9.8 with

1% PEG 80Θ0 and 10 mM MgCtø. The results appear as distinct blue-black bands.

Example 6

Dipstick tests for urinary tract infections have been developed for determining the presence of the bacteria

Escherichia coli, Proteus, Klebsiella, Enterobacter, Enterococci and Staphylococcus.

Antibodies against each of these bacteria are prepared in individual goats by injections once a week over 1 month. The bacteria are administered in Freund's adjuvant containing heat killed bacteria (70° C for 45 minutes) as the immugen. Serums from the goats are collected and pooled over a length of time and then affinity purified using an immunoadsorbant column prepared by immobilizing the specific bacterial cells in an agarose matrix or antigens or antigen substrate bound or conjugated to a solid chromatographic support. U actual antigen or cell affinity purification is not required for the sensitivity of the assay, then classical ammonium sulfate precipitation or molecular exchange chromatography may b used as well as isolation of IgG fractions using protein A or protein G columns. In this example, the polyclonal antibodies are against the flagellar and somatic antigens found on the cell wall and cell membranes of the bacteria.

The polyclonal antibody against these bacteria are bound in a linear or any other distinguishable patterns to a film like carrier member on a dipstick. The carrier member contains one positive control zone and 6 specimen reaction zones against the six bacteria. The polyclonal antibodies against these bacteria are bound in a linear and other pattems to distinguish one from another. The antibodies are applied at a concentration of 1.5 μg/5 mm of nylon carrier. The antibody patterns are applied to the nylon carrier using a Camag TLC spotter device (Camag Scientific Inc., Wilmington, NC).

After the reagents dry, the earner membrane is immersed in 100% goat serum for 2 minutes, immediately followed by immersion in 0.3% casein in 0.015 M phosphate buffer at a pH of 7.4. The carrier membrane is allowed to dry for 20 minutes in less then 25% relative humidity conditions and then immersed in a 1% mannitol solution in 0.1 M borate buffer, pH 8.4.

Specific polyclonal conjugates wherein the antibodies are bound to alkaline phosphatase are prepared using the glutaraldehyde procedure. To minimize background color due to free or unbound phosphatase, the conjugates are purified by one of the following methods: a) dialysis; b) membrane concentration devices; c) sephadex molecular exchange chromatography.

Urine samples are mixed with polyclonal antibody to which is bound alkaline phosphatase in sample test vials at room temperature. After 2 minutes, the dipstick is inserted into the reaction mixture in the test vials and permitted to incubate for 10 minutes. Without any additional washing steps, the dipsticks are then placed in the dye solution containing 0.8 g/1 BCIP, 0.3 g/1 NBT in 0.1 M AMP, pH 9.8 with 1% PEG 8000 and 10 mM

MgC for three to five minutes. The positive control zone develops color as a dark blue-black line. If the various antigens are present, the appropriate specimen reaction zone patterns would appear. No quenching steps are required at the end of the assay and the dipsticks can be stored as a permanent record.

Example 7

A variation of the dipstick protocol of Example 6 involves the placement of the antibodies on two separate dipsticks,

34

each detecting three analytes and a positive control reference zone. The two separate dipsticks may be attached at the end opposite to the carrier support to form a "wishbone" configuration or kept separate. The test are run in a similar fashion as described in Example 6, except that the dipsticks are placed in their corresponding vials containing the appropriate polyclonal antibodies conjugated to alkaline phosphatase and incubated together in the dye tube.

Example 8 This is an example of binding an antigen to the solid carrier member and assaying for an antibody in the body fluid. An assay for detecting infectious mononucleosis heterophile antibody has been developed by adsorbing beef cell antigen onto the carrier member of the dipstick. The infectious mononucleosis heterophile antigen on bovine red cells bind specifically to the IM heterophile IgM antibody. The beef cell antigen is extracted from beef cell erythrocytes and is then bound in a linear pattern on the dipstick and blocked as described hereinabove. To perform the assay, the dipstick is inserted into the serum sample for two to three minutes and then transferred into a second vessel containing antibody against human IgM which is bound to alkaline phosphatase. The dipstick is incubated for two to three minutes at room temperature. The dipstick is then transferred into the dye solution and the results observed after three minutes. Example 9

Serum antinuclear antibodies are associated with systemic lupus erythematosus (sLE) and the antibodies associated with sLE are against deoxyribonucleic acid protein. A systemic lupus erythematosus diagnostic test is performed by adsorbing deoxyribonucleic acid protein onto the carrier member as the bound capture reagent. The test is then run in a manner similar to Example 8. Example 10

A competitive dipstick assay system for digoxin and theophylline are performed where the specific antigens are bound to the carrier support on the dipstick and the carrier support is blocked. The sample is admixed with the antibody to which is bound the alkaline phosphatase and incubated for up to five minutes. The dipstick is then placed in the sample test vial. For negative samples, unbound antibody conjugate will be bound to the dipstick. For positive samples, i.e., those samples with analyte present, the analyte will bind to the antibody, thereby preventing the antibody from binding to the dipstick. After the incubation period, the dipstick is transferred to the dye or substrate vial. The color formed will be indirectly proportional to the amount of specific antigen present ^"n the sample. The digoxin assay is developed to give a dt β response curve of between 0 and approximately 5.0 ng/ml. The preparation of a dose response curve is well known to those of ordinary skill in the art. The theophylline assay has a dose response curve of between 0 and approximately 40 μg/ml of serum.

Although assays for human chorionic gonadotropin,

Salmonella analyte, or bacterial antigens as an analyte have been described, it will be appreciated that a similar assay for other analytes may be employed in accordance with the invention. Other such analytes that may be assayed include Epstein Barr, chlamydia, lymes disease, Proteus, Klebsiella, Candida

Staphlococcus, Pseudomonas, Clostridium difficile, hepatitis A and B, human immunodeficiency virus (HIV), Equine pregnancy, canine heartworm, feline leukemia etc. If a label other than an enzyme or color development materials other than dye solutions are used, the procedure may be varied, but these and other such variations by persons skilled in the art may be made without departure from the spirit of the invention. Furthermore, while the invention has been described as being applicable to the medical field for detecting the presence of substances in various

Claims

WHAT ^CLAIMED IS:

1. An immunoassay process for determining whether a target immunologically active agent is present in a fluid sample, comprising the steps of: a. contacting a fluid sample with a labeled capture reagent against a target immunologically active agent to be assayed; b. contacting the fluid sample and the labeled capture reagent with a carrier member having bound to a surface thereof an effective amount of a bound capture reagent against the target immunologically active agent; and c. determining whether the labeled capture reagent is bound to the solid carrier, the quantity of the labeled capture reagent which binds to the carrier member indicating the quantity of the target immunologically active agent in the fluid sample

2. The process of Claim 1, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member having bound to a surface thereof an effective amount of a bound capture reagent against the target immunologically active agent comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution, whereby the carrier member is blocked against non-specific binding by the labeled capture reagent

3. The process of Claim 2, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, then dried prior to being treated with a buffer solution.

4. The process of Claim 1, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member having bound to a surface thereof an effective amount of a bound capture reagent against the target immunologically active agent comprises the step of contacting the fluid sample and the labeled capture reagent with a canier member which has been treated with a solution of an effective amount of serum and and effective amount of casein and then treated with a buffer solution, whereby the carrier member is blocked against non-specific binding by the labeled capture reagent.

5. The process of Claim 1, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member having bound to a surface thereof an effective amount of a bound capture reagent against the target immunologically active agent comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, treated with a buffer solution, and then dried.

6. The process of Claim 2, wherein the step of contacting the fluid sample and the labeled capture reagent with the carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum selected from the group consisting of goat serum, pig serum, bovine serum, fetal calf serum, mouse serum, calf serum, rabbit serum, and human serum, the solid carrier thereafter being treated with an effective amount of a solution of a protein and then treated with a buffer solution.

7. The process of Claim 2, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a canier member which has been treated with an effective amount of goat serum, the solid carrier thereafter being treated with an effective amount of a solution of a protein and then treated with a buffer solution.

8. The process of Claim 2, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent witfi a canier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of milk protein, and then treated with a buffer solution.

9. The process of Claim 8, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of milk protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a canier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of casein, and then treated with a buffer solution.

10. The process of Claim 9, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of casein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of Hammarsten casein, and then treated with a buffer solution.

11. The process of Claim 2, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution selected from the group consisting of borate buffer, hepes buffer, phosphate buffer, tris buffer, and pipes buffer.

12. The process of Claim 11, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a borate buffer.

13. The process of Claim 2, wherein the step of contacting the fluid sample and the labeled capture reagent with a canier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of contacting the fluid sample and the labeled capture reagent with a canier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution containing an effective amount of a sugar selected from the group consisting of mannitol, sucrose, sorbitol, corn syrup, glucopyranosides, maltose, fructose, glucose, xylose, and oligosaccharides.

14. The process of Claim 13, wherein the step of contacting the fluid sample and the labeled capture reagent with a carrier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution comprises the step of '. , itacting the fluid sample and the labeled capture reagent with a canier member which has been treated with an effective amount of serum, treated with an effective amount of a solution of a protein, and then treated with a buffer solution containing an effective amount of mannitol.

15. The process of Claim 1, wherein the carrier member is comprised of a material selected from the group consisting of latex, polyethylene, nylon, nylon film, polypropylene, ethylenepropylene copolymers, polybutylenes, polystyrene, polyvinyl chloride, polyvinylidene chloride and polytetrafluoroethylene, polyethylene terephthalate, polyacrylate polymethyl-acrylate, cellulose, fibrous cellulose, glass, and glass fibers.

16. The process of Claim 15, wherein the carrier member is comprised of nylon film.

17. The process of Claim 1, wherein the canier member comprises a plurality of solid particles.

18. The process of Claim 1, wherein the bound capture reagent against the target immunologically active agent is bound to a surface of the carrier member at a concentration of at least approximately 0.03 μg/mm².

19. The process of Claim 18, wherein the bound capture reagent comprises an antibody.

20. The process of Claim 18, wherein the bound capture reagent comprises an analyte.

21. The process of Claim 1, wherein the target immunologically active agent comprises an analyte.

22. The process of Claim 21, wherein the labeled capture reagent is an enzyme labeled antibody against the analyte.

23. The immunoassay process of Claim 1, wherein the labeled capture reagent and the bound capture reagent comprise monoclonal or polyclonal antibodies or mixtures thereof.

24. The immunoassay process of Claim 1, wherein the fluid sample is selected from the group consisting of body fluid, culture media, food, and water.

25. The immunoassay process of Claim 1, wherein the labeled capture reagent comprises an enzyme-labeled antibody.

26. The immunoassay process of Claim 1, wherein the step of determining whether the labeled capture reagent is bound to the solid carrier comprises the step of contacting the carrier member with a reactant that will react with the labeled capture reagent to generating a signal.

27. The immunoassay process of Claim 26, wherein the reactant comprises an enzyme substrate.

28. The process of Claim 27, wherein the enzyme substrate that will react with the enzyme is in a time-release form in an admixture of the fluid sample of target immunologically active agent and the labeled capture reagent.

29. The process of Claim 1, wherein the target immunologically active agent is a hormone.

30. The process of Claim 1, wherein the target immunologically active agent is an antigen associated with a microorganism causing a urinary tract infection.

31. The process of Claim 30, wherein the antigen associated with a microorganism causing a urinary tract infection is selected from the group comprising Enterobacter sp., Enterococcus, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Proteus sp., Staphylococcus epidermidis (albus), α-hemolytic streptococcus, and β-hemolytic streptococcus.

32. The process of Claim 1, wherein the target immufiologically active agent is a bacterial antigen.

33. The process of Claim 1, wherein the target immunologically active agent is a viral antigen.

34. An apparatus for use in an immunoassay process for the detection of a target immunologically active agent in a fluid sample, comprising: a. a carrier member to which is bound an effective amount of a capture reagent to a target immunologically active agent in a fluid sample; and b. at least one substantially non-absorbent container inert to immunologically active agents in which an admixture of immunologically active agents can be reacted and into which the carrier member can be inserted in contact with the admixture of active agents.

35. The apparatus of Claim 34, wherein the carrier member comprises a carrier member which has been treated with an effective amount of serum, then treated with an effective amount of a solution of a protein, and then treated with a buffer solution, whereby the carrier member is blocked so that the labeled capture reagent will not non-specifically bind to the canier member.

36. The apparatus of Claim 35, wherein the carrier member is dried after being treated with an effective amount of the solution of a protein and prior to being treated with the buffer solution.

37. The apparatus of Claim 34, wherein the carrier member comprises a carrier member which has been treated with a solution with an effective amount of serum and an effective amount a protein, and then treated with a buffer solution, whereby the carrier member is blocked so that the labeled capture reagent will not non-specifically bind to the carrier member.

38. The apparatus of Claim 35, wherein the carrier member, after having been treated with the buffer solution, has been dried.

39. The apparatus of Claim 34, wherein the capture reagent is bound to the carrier member at a concentration of at least 0.03 μg/mm².

40. The apparatus of Claim 34, wherein the bound capture reagent comprises an antibody.

41. The apparatus of Claim 34, wherein the bound capture reagent comprises an analyte.

42. The apparatus of Claim 35, wherein the serum is selected from the group consisting of goat serum, pig serum, bovine serum, fetal calf serum, calf serum, mouse serum, rabbit serum and human serum.

43. The apparatus of Claim 42, wherein the serum comprises goat serum.

44. The apparatus of Claim 35, wherein the protein comprises a milk protein.

45. The apparatus of Claim 44, wherein the milk protein comprises casein.

46. The apparatus of Claim 45, wherein the casein comprises Hammarsten casein.

47. The apparatus of Claim 35, wherein the buffer solution is selected from the group consisting of borate buffer, hepes buffer, phosphate buffer, tris buffer, and pipes buffer.

48. The apparatus of Claim 47, wherein the buffer solution comprises a borate buffer.

49. The apparatus of Claim 35, wherein the buffer solution contains a sugar which is selected from the group consisting of mannitol, sucrose, sorbitol, corn syrup glucopyranosides, maltose, fructose, glucose, xylose, and oligosaccharides.

50. The apparatus of Claim 49, wherein the sugar comprises mannitol.

51. The apparatus of Claim 34, wherein the canier member is selected from the group consisting of latex, polyethylene, nylon, nylon film, polypropylene, ethylenepropylene copolymers, polybutylenes, polystyrene, polyvinyl chloride, polyvinylidene chloride and polytetrafluoroethylene, polyethylene terephthalate, polyacrylate polymethyl-acrylate, cellulose, fibrous cellulose, glass and glass fibers.

52. The apparatus of Claim 51, wherein the carrier member comprises nylon film.

53. The apparatus of Claim 34, wherein the canier member comprises a plurality of particles.

54. The apparatus of Claim 34, wherein the target immunologically active agent comprises an analyte.

55. The apparatus of Claim 34, wherein the bound capture reagent comprises a polyclonal antibody, a monoclonal antibody or a mixture thereof.

56. The apparatus of Claim 34, wherein the bound capture reagent is bound over only a portion of one surface of the canier member in a band.

57. The apparatus of Claim 34, wherein the canier member comprises opposing first and second surfaces, wherein said capture reagent is bound to said first surface, and further comprising a handle attached to said second surface.

58. The apparatus of Claim 34, wherein the canier member is comprised of a polymeric material.

59. The apparatus of Claim 34, wherein the target immunologically active agent is a hormone.

60. The apparatus of Claim 34, wherein the target immunologically active agent is an antigen associated with a microorganism causing a urinary tract infection.

61. The apparatus of Claim 60, wherein the antigen associated with a microorganism causing a urinary tract infection is selected from the group comprising Enterobacter sp., Enterococcus, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Proteus sp., Staphylococcus epidermidis (albus), α-hemolytic streptococcus, and β-hemolytic streptococcus

62. The apparatus of Claim 34, wherein the target immun Lologically active agent is a bacterial antigen.

63. The apparatus of Claim 34, wherein the target immunologically active agent is a viral antigen.

64. A dip-stick test device for use in an immunoassay for the detection of a target immunologically active agent in a fluid sample, the device comprising: a. a carrier member having a first and second surface; b. an effective amount of a capture reagent against a target immimologically active agent in a fluid sample, the capture reagent being bound to said first surface; and c. a handle attached to said second surface by which the carrier member may be handled.

65. The dip-stick test device of Claim 64, wherein the bound capture reagent is bound to the carrier member at a concentration of at least approximately 0.03 μg/mm².

66. The dip-stick test device of Claim 64, wherein the bound capture reagent comprises an antibody.

67. The dip-stick test device of Claim 64, wherein the bound capture reagent comprises an analyte.

68. The dip-stick test device of Claim 64, wherein the carrier member comprises a polymeric material.

69. The dip-stick test device of Claim 64, wherein the capture reagent is bound to only a portion of said first surface.

70. The dip-stick test device of Claim 64, wherein the bound capture reagent is chemically bound to the carrier member.

71. The dip-stick test device of Claim 64, wherein the canier member comprises a thin film of polymeric material to which the capture reagent may be bound.

72. The dip-stick test device of Claim 64, wherein more than one capture reagent is bound to the first surface of the carrier member, each bound capture reagent being bound to the carrier membrane in a separate region.

73. The dip stick test device of Claim 64, wherein the carrier member comprises a carrier member which has been treated with an effective amount of serum, then treated with an effective amount of a solution of a protein, and then treated with a buffer solution, whereby the carrier member is blocked so that the labeled capture reagent will not non-specifically bind to the canier member.

74. The dip-stick device of Claim 73, wherein the carrier member is dried after being treated with an effective amount of the solution of a protein and prior to being treated with the buffer solution.

75. The dip-stick test device of Claim 73, wherein the carrier, member, after having been treated with the buffer solution, ha been dried.

76. The dip-stick test device of Claim 73, wherein the serum is selected from the group consisting of goat serum, pig serum, bovine serum, fetal calf serum, calf serum, mouse serum, rabbit serum, and human serum.

77. The dip-stick test device of Claim 76, wherein the serum comprises goat serum.

78. The dip-stick test device of Claim 73, wherein the protein comprises a milk protein.

79. The dip-stick test device of Claim 78, wherein the milk protein comprises casein.

80. The dip-stick test device of Claim 79, wherein the casein comprises Hammarsten casein.

81. The dip-stick test device of Claim 73, wherein the buffer solution is selected from the group consisting of borate buffer, hepes buffer, phosphate buffer, tris buffer, and pipes buffer.

82. The dip-stick test device of Claim 81, wherein the buffer solution comprises a borate buffer.

83. The dip-stick test device of Claim 73, wherein the buffer solution contains a sugar which is selected from the group consisting of mannitol, sucrose, sorbitol, corn syrup glucopyranosides, maltose, fructose, glucose, xylose, and oligosaccharides.

84. The dip-stick test device of Claim 83, wherein the sugar is mannitol.

85. The dip-stick test device of Claim 64, wherein the carrier member is comprised of a material which is selected from the group consisting of latex, polyethylene, nylon, nylon film, polypropylene, ethylenepropylene copolymers, polybutylenes, polystyrene, polyvinyl chloride, polyvinylidene chloride and polytetrafluoroethylene, polyethylene terephthalate, polyacrylate polymethyl-acrylate, cellulose, fibrous cellulose, glass and glass fibers.

86. The dip-stick test device of Claim 85, wherein the carrier member is nylon film.

87. The dip stick device of Claim 64, wherein the target immunologically active agent is a hormone.

88. The dip stick device of Claim 64, wherein the target immunologically active agent is an antigen associated with a microorganism causing a urinary tract infection.

89. The dip stick device of Claim 88, wherein the antigen associated with a microorganism causing a urinary tract infection is selected from the group comprising Enterobacter sp.,

Enterococcus, Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Proteus sp., Staphylococcus epidermidis (albus), α-hemolytic streptococcus, and β-hemolytic streptococcus

90. The dip stick device of Claim 64, wherein the target immunologically active agent is a bacterial antigen.

91. The dip stick device of Claim 64, wherein the target immunologically active agent is a viral antigen.

92. A process of preparing a carrier member for use in an immunoassay that utilizes the canier member to separate an immunologic complex from a reaction solution, the process comprising the step of binding an effective concentration of an immunologically active agent to the surface of the canier member.

93. The process of Claim 92, wherein the immunologically active agent is bound to the carrier member at a concentration of at least 0.03 μg of immunologically active agent per mm².

94. The process of Claim 92, wherein the immunologically active agent comprises an antibody.

95. The process of Claim 92, wherein the immunologically active agent comprises an analyte.

96. The process of Claim 92, comprising the further steps of: a. treating the carrier member with an effective amount of serum; b. then treating the carrier member with an effective amount of a solution of a protein; and c. treating the solid carrier with a buffer solution.

97. The process of Claim 96, further comprising the step of drying the carrier member after step (b) and before step (c).

98. The process of Claim 96, wherein the serum is selected from the group consisting of goat serum, pig serum, bovine serum, fetal calf serum, calf serum, mouse serum, rabbit serum and human serum.

99. The process of Claim 98, wherein the serum comprises goat serum.

100. The process of Claim 96, wherein the protein comprises a milk protein.

101. The process of Claim 100, wherein the milk protein comprises casein.

102. The process of Claim 101, wherein the casein comprises Hammarsten casein.

103. The process of Claim 96, wherein the buffer solution is selected from the group consisting of borate buffer, hepes buffer, phosphate buffer, tris buffer and pipes buffer.

104. The process of Claim 103, wherein the buffer solution comprises a borate buffer.

105. The process of Claim 96, wherein the buffer solution contains a which sugar is selected from the group consisting of mannitol, sucrose, sorbitol, corn syrup glucopyranosides, maltose, fructose, glucose, xylose, and oligosaccharides.

106. The process of Claim 105, wherein the sugar comprises mannitol.

107. The process of Claim 96, further comprising the step of drying the carrier member after step (c). ^~

108. The process of Qaim 92, wherein the carrier member is comprised of a material which selected from the group consisting of latex, polyethylene, nylon, nylon film, polypropylene, ethylenepropylene copolymers, polybutylenes, polystyrene, polyvinyl chloride, polyvinylidene chloride and polytetrafluoroethylene, polyethylene terephthalate, polyacrylate polymethyl-acrylate, cellulose, fibrous cellulose, glass and glass fibers.

109. The process of Claim 108, wherein the carrier member comprises nylon film.

110. The process of Qaim 92, wherein the carrier member comprises a plurality of solid particles.

111. A solid phase component of an immunoassay that utilizes die carrier member to separate an immunologic complex from a reaction solution, the carrier member comprising an insoluble solid material to which an effective amount of an immunologically active agent has been bound.

112. The solid phase component of Claim 111, wherein the immunologically active agent is bound to the carrier member at a concentration of at least approximately 0.03 μg of immunologically active agent per mm².

113. The solid phase component of Claim 111, wherein the immunologically active agent comprises an antibody.

114. The solid phase component of Claim 111, wherein the immunologically active agent comprises an analyte.

115. A solid phase component of an immunoassay being manufactured by a process comprising the steps of: a. bonding the immunologically active agent to a substrate; b. treating the substrate with an effective amount of serum; c. then treating the substrate with an effective amount of a solution of a protein; and d. treating the substrate with a buffer solution.

116. The component of Claim 115, which is manufactured by a process comprising the further step of drying the carrier member after step (b) and before step (c).

117. The component of Claim 115, wherein in step (b), the serum is selected from the group consisting of goat serum, pig serum, bovine serum, mouse serum, fetal calf serum, calf serum, rabbit serum and human serum.

118. The component of Claim 117, wherein in step (b), the serum comprises goat serum.

119. The component of Claim 115, wherein in step (c), the protein comprises a milk protein.

120. The component of Claim 119, wherein in step (c), the milk protein comprises casein.

121. The component of Claim 120, wherein in step (c), the casein is Hammarsten casein.

122. The component of Claim 115, wherein in step

(d), the buffer solution is selected from the group consisting of borate buffer, hepes buffer, phosphate buffer, tris buffer and pipes buffer.

123. The component of Claim 122, wherein in step

(d), the buffer solution comprises a borate buffer.

124. The component of Claim 115, wherein in step

(d), the buffer solution contains a sugar is selected from the group consisting of mannitol, sucrose, sorbitol, corn syrup glucopyranosides, maltose, fructose, glucose, xylose, and oligosaccharides.

125. The component of Claim 124, wherein in step (d), the sugar comprises mannitol.

126. The component of Claim 115, wherein the substrate is comprised of a material selected from the group consisting of latex, polyethylene, nylon, nylon film, polypropylene, ethylenepropylene copolymers, polybutylenes, polystyrene, polyvinyl chloride, polyvinylidene chloride and polytetrafluoroethylene, polyethylene terephthalate, polyacrylate polymethyl-acrylate, cellulose, fibrous cellulose, glass and glass fibers.

127. The component of Claim 126, wherein the substrate comprises nylon film.

128. The component of Claim 115, wherein the substrate comprises a plurality of solid particles.