WO1994016329A1

WO1994016329A1 - Methods for solid phase capture in immunoassays

Info

Publication number: WO1994016329A1
Application number: PCT/US1994/000429
Authority: WO
Inventors: Jhan C. Swanson; Joy C. Sonsalla; Jeng Tzyy-Wen
Original assignee: Abbott Laboratories
Priority date: 1993-01-13
Filing date: 1994-01-12
Publication date: 1994-07-21
Also published as: AU5994294A

Abstract

A method for the determination of the presence or amount of an analyte by monitoring a detectable label immobilized within an asymmetric membrane. A test mixture, comprising a test sample, an indicator reagent attached to a first specific binding pair member, and a capture reagent attached to a second specific binding pair member immobilized on a solid phase, is formed which is then captured within the asymmetric membrane thereby enabling analyte concentration to be determined.

Description

METHODS FOR SOLID PHASE CAPTURE IN IMMUNOASSAYS

BACKGROUND OF THE INVENTION

1 . Field of Invention:

This invention relates to a method for measuring analytes in a liquid medium. More specifically, this invention relates to a method for performing solid phase immunoassays using electrochemical detection methods. It also relates to a method for immobilizing microparticles useful in performing immunoassays independent of detection method.

2. Description of the Prior Art: Immunoassays are a specific type of receptor ligand assay which have been useful in measuring a wide variety of analytes in liquid medium, where receptor and ligand are specific binding pair members which demonstrate a high and specific affinity for each other. These assays require, in general, a method for measuring either the amount of binding pair member which is free in solution or the amount which is bound to its corresponding binding pair member at the conclusion of the assay reactions. Solid phase immunoassays have been been found to be especially useful for separation of bound and free binding pair members. In general, the solid phase will have attached thereto a member of a specific binding pair member with a detectable label directly or indirectly attached thereto, the amount of detectable label being related to the concentration of analyte in solution. In these methods, one of the binding pair members is in some way immobilized on the same or another solid phase, and a physical wash step is incorporated into the assay protocol to remove excess free binding pair member. The amount of binding pair member either immobilized on the solid phase or free in solution, can then be measured directly.

Microparticles have been found to be especially useful as a solid phase in immunoassays. The particles can be easily suspended in solution and have a large surface area which facilitates the reaction of the solid phase bound specific binding pair member with solution components. The use of microparticles has been described in the prior art. U.S. Patent 4,517,288 (Giegel et al.) describes a method for performing a ligand assay in an inert porous medium by immobilizing a binding material within a finite zone of the medium, subsequently applying analyte and labeled indicator to the medium, separating unbound labeled indicator by applying a stream of solvent sufficient to effect radial chromatographic separation, and determining the amount of labeled indicator remaining in the zone. U.S. Patent 4,752,562 (Sheiman et al.) describes a similar method for measurement of microbial antigens. The porosity of the seperation matrix medium is important to capture particles of microbial size. The matrix is chosen so that the interstitial space size is sufficiently small enough to trap the microbial sized particles. U.S. Patent 4,552,839 (Gould et al.) describes analyte detection in a particle containing medium by contacting the medium with a bibulous material , wherein particles are concentrated at a position adjacent to the contact site by wicking of particles past the position and a signal associated with these particles can be used to measure the presence of analyte. U.S. Patent 4,666,863 (Edwards et. al.) describes a method for performing an immunoassay where analyte in the sample is mixed with labeled reagents prior to applying the mixture on the chromatographic medium. The label is present in two forms which are either mobile or immobile on the chromatographic medium depending on the analyte concentration. The further the mobile species migrates radially on the chromatographic medium, the easier to analyze either the mobile or immobile species. U.S. Patent 4,652,533 (Jolley) describes a solid phase immunoassay where particles are reacted with an analyte while in a suspended state and are then concentrated prior to or subsequent to reaction with a luminescent label. European Patent Application 0 422 699 A2 (Calderhead et. al.) describes a method for immobilizing particles on a bibulous material where the particles are small enough to infiltrate the pores of the material but large enough to prevent the particles from diffusing from the pores.

The binding pair member may be immobilized on the solid phase directly, or may be immobilized on the solid phase by conjugation to a member of a second specific binding pair, where the solid phase contains the complementary second binding pair member. This type of indirect capture has the advantage that the interaction of specific binding pair members involving the analyte of interest occurs in the solution phase and that it is possible to use a single type of solid phase for performing immunoassays on multiple analytes. An example of this type of scheme is described in WO 90/04786 (Olson et al.). This patent application describes a porous capture membrane for removing immunocomplexes from solution, where the solid phase carries a hapten such as biotin and the binding pair member for the analyte of interest is conjugated to an anti-hapten component (streptavidin). This type of capture system has been utilized by the Threshold™ system manufactured by Molecular Devices, Inc. (Menlo Park, CA ). This system uses an electrochemical method to detect enzyme activity immobilized on a solid microporous membrane. For electrochemical detection methods it is essential that the analyte be in electrical contact with the sensor. These methods will therefore also require in general that the detectable label be non-diffusively associated with the solid phase support.

There are a number of practical issues which need to be addressed when designing an immunoassay. The detection method must be sufficiently sensitive to detect the labeled binding pair member at the concentrations of interest, and should be able to detect the signal reproducibly with minimal random error caused by assay delivery format. The measurement of signal from a given sample should not contaminate or otherwise affect readings of subsequent samples. Furthermore, the interaction of binding pair members and the capture of detectable label on the solid phase should not be subject to interfering components in the sample. The label should be non-diffusively associated with the solid support to allow washing away of unbound label and other manipulations as appropriate. This is necessary for electrochemical detection methods where the solid phase needs to be immersed in solution so it is in electrical contact with the sensor. It is also necessary when chromatographic or wicking mechanisms are used to separate bound and free binding pair members. It is preferable that the solid phase be low in cost. Normal variability in the manufacturing process used to produce the solid support should not cause errors in accurate detection of the analyte of interest.

For both optical and electrochemical detection methods, the chemical capture system described above suffers from the limitation that capture of binding pair members on the solid support is subject to chemical interferences from substances present in serum. Furthermore, the preparation of the microporous membrane with associated binding pair member is an expensive process subject to manufacturing variabilities which can adversely affect measurement of the analyte of interest. Microparticle capture does not suffer from these limitations, but its use with electrochemical methods has been limited for other reasons. Microparticles immobilized on the surface of a microporous membrane fall off or disassociate themselves from the membrane when the microporous membrane is immersed in solution and also have been found to rub off on to the detector causing sensor contamination. Microparticle capture methods have been used with luminescent detection methods, but also suffer limitations. Optical detection methods with particles are sensitive to capture geometry as particles must be in optical contact with the detector unit. This limits the number of particles which can be concentrated by capture within a given area of membrane. Optical detection methods will in general be sensitive to random error caused by variations in optical properties of the membrane and capture geometry. When chromatographic or wicking mechanisms are used to separate bound and free binding pair members, microparticles again suffer from the limitation that they are not fully immobilized within the membrane.

It is an object of the current invention to describe a method for performing immunoassays using microparticles as a solid support with electrochemical detection of bound label which does not suffer the limitations of chemical capture methods and which obviates the disadvantages of the current art involving microparticles. It is also an object of the current invention to describe an improved detection method for performing immunoassays where binding pair members are immobilized on microparticles which are further immobilized on a microporous membrane which eliminates the limitiations of optical detection methods. It is also an object of the current invention to provide a method for immobilizing microparticles non-diffusively on microporous membrane supports in such a manner that they remain associated with the membrane after suspension in buffer solutions. SUMMARY OF THE INVENTION

The present invention relates to a method for determining the presence or amount of an analyte in a test sample. The test sample is contacted either sequentially or simutaneously with an indicator reagent and a capture reagent. The indicator reagent comprises a first specific binding pair member directly or indirectly attached to a detectable label. The capture reagent comprises a second specific binding pair member directly or indirectly attached to a microparticle. The analyte binds to one of the binding pair members and analyte detection occurs by immobilizing microparticles on an asymmetric membrane where one side of the membrane has a pore size sufficiently large to allow the microparticles to enter and a second side which has pores of a size sufficiently small to prohibit microparticles from passing through. The membrane is submerged in a liquid medium and read in either a verticle or inverted position.

DETAILED DESCRIPTION OF THE INVENTION

A "specific binding member", as used herein , is a member of a specific binding pair, i.e., two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. In addition to antigen and antibody-specific binding pairs, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences (including probe and nucleic acid sequences used in DNA hybridization assays to detect a target nucleic acid sequence), complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding member. For example, a derivative or fragment of the analyte, i.e., an analyte-analog, can be used so long as it has at least one epitope in common with the analyte. Immunoreactive specific binding members include antigens, haptens, antibodies, and complexes thereof including those formed by recombinant DNA methods or peptide synthesis.

"Analyte", as used herein, is the substance to be detected in the test sample using the present invention. The analyte can be any substance for which there exists a naturally occurring specific binding pair member (e.g., an antibody) or for which a specific binding pair member can be prepared, and the analyte can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances, haptens, antibodies, or combinations thereof. The analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.

"Capture Reagent," as used herein, is a specific binding member, capable of binding the analyte or indicator reagent, which can be directly or indirectly attached to a substantially solid material. The solid phase capture reagent complex can be used to separate the bound and unbound components of the assay.

"Indicator reagent," as used herein comprises a detectable label directly or indirectly attached to a specific binding member or metal surface.

"Test mixture," as used herein, means a mixture of the test sample and other substances used to apply the present invention for the detection of analyte in the test sample. Examples of these substances include: Specific binding members, ancillary binding members , analyte-analogs, buffers, and diluents.

"Test sample," as used herein, means the sample containing the analyte to be detected and assayed using the present invention. The test sample can contain other components besides the analyte, can have the physical attributes of a liquid, or a solid, and can be of any size or volume, including for example, a moving stream of liquid. The test sample can contain any substances other than the analyte as long as the other substance do not interfere with the specific binding of the specific binding member or with the analyte or the analyte-analog. Examples of test samples include, but are not limited to: Serum, plasma, sputum, seminal fluid, urine, other body fluids, and environmental samples such as ground water or waste water, soil extracts and pesticide residues.

"Analyte-analog," as used herein, refers to a substance which cross reacts with an analyte specific binding member although it may do so to a greater or lesser extent than does the analyte itself. The analyte-analog can include a modified analyte as well as a fragmented or synthetic portion of the analyte molecule so long as the analyte analog has at least one epitopic site in common with the analyte of interest.

"Analyte-mediated ligand binding event," as used herein, means a specific binding event between two members of a specific ligand binding pair, the extent of the binding is influenced by the presence, and the amount present, of the analyte. This influence usually occurs because the analyte contains a structure, or epitope, similar to or identical to the structure or epitode contained by one member of the specific ligand binding pair, the recognition of which by the other member of the specific ligand binding pair results in the specific binding event. As a result, the analyte specifically binds to one member of the specific ligand binding pair, thereby preventing it from binding to the other member of the specific ligand binding pair.

"Ancillary specific binding member," as used herein, is a specific binding member used in addition to the specific binding members of the captured reagent and the indicator reagent and becomes a part of the final binding complex. One or more ancillary specific binding members can be used in an assay. For example, an ancillary specific binding member can be used in an assay where the indicator reagent is capable of binding the ancillary specific binding member which in turn is capable of binding the analyte.

"Enhancer," as used herein, is any substance which, when present in the test mixture, facilitates a binding, an association, or an agglutination event among particles or soluble substances in a solution or suspension. Enhancers function by changing the pH, ionic, solvent or colligative properties of the liquid medium, or in other ways. Examples of enhancers include, but are not limited to: Salts, such as sodium chloride; any type of buffer preparation which would serve to maintain a desired pH; sugars; and polymers, such as polyethylene glycol.

The quantitation of analyte in liquid samples, according to the current invention, requires microparticles, an asymmetric membrane for capture of particles, electrochemical or other detection apparatus, and reagents. The membranes will be asymmetric membranes having a larger pore size on one the side of the membrane, and a smaller pore size on the opposing side of the membrane. Membranes useful in this respect will generally be of two types. The first type are supported microporous membranes. These membranes are typically made of nitrocellulose, polysulfone, nylon, polyvinylidene fluoride (PDVF), polytetrafluoroethylene (PTFE) or other microporous membranes with a support which may be made of polypropylene, polyester or other material. It is believed that the support should have a pore size which is sufficiently large to allow the microparticles to enter the membrane. It is also believed that the microporous membrane should have a pore size sufficiently small to restrict microparticles from entering or otherwise passing completely through the membrane. The microporous membrane will therefore have a pore size which will be smaller than the pore size of the fabric support. The pore size of the microporous membrane should typically range between 0.1 and 5μ. An example of this type of membrane is Gelman SV-450 (Gelman Sciences, Ann Arbor, Mi., 48106) consisting of a 0.45μ polysulfone membrane on a non-woven polypropylene support. Other membranes of this type are described in the examples below. The above conditions are those generally believed to be necessary for satisfactory performance. It will be seen from the examples below that not all supported membranes will be suitable for use is acordance with the current invention. However, those supported membranes found to be useful exhibit the aforementioned asymmetric properties.

The second type of membrane are those where the pore size continuously changes from one side of the membrane to the other. A useful example of this type of membrane is Filterite BTS30 asymmetric polysulfone membrane (Memtek America Corporation, DeLand, FL., 32724-9990) where the pore size decreases from 45μ on one side of the membrane to 0.45μ on the other side of the membrane.

The microparticles should generally be less than 20 μM in diameter, and should be of such a size that they are able to enter the membrane on the more porous side but are unable to pass through the membrane. The particles will be water insoluble, and can be naturally occurring materials such as cells or fragments thereof, or be made from synthetic materials. Binding pair members can be attached to these particles by adsorption, covalent or other means of direct or indirect attachment. It is necessary that the binding pair members remain associated with the particles and be able to bind to their corresponding binding pair member. It is also necessary that the particles have low non-specific interactions with other substances in the sample and binding pair members.

The particles are captured or immobilized on the membrane by applying a solution of the particles to the more porous side of the membrane and causing the particles to be drawn into the depth of the membrane. Some possible means of drawing the particles into the membrane include pressure differentials across the membrane and movement of solution due to capillary flow. The particles immobilized within the depth of the membrane may be reacted with various assay components or washed as described below.

The particles immobilized within the membrane will generally contain, directly or indirectly, a detectable label which can be related to the concentration of analyte initially present in the sample. The label may be directly detectable by a suitable sensing system, such as detection of fluorophore by fluorescent methods, or may be an enzyme, catalyst , or other amplification scheme which causes a change in an associated substance which can be detected such as detection of methylumbelliferyl phosphate hydrolysis by alkaline phosphatase which can be monitored by fluorescent methods. Rate or endpoint methods may also be utilized.

Any suitable detection methods may be used. Detection methods involving measurement of electromagnetic radiation will include methods such as absorbance, fluorescence, fluorescence polarization, visual appearance. When using surface detection methods such as surface reflectance and surface fluorescence, it will be important that the particles be immobilized in such a manner that they are at least partially visible from the detection side of the membrane.

Especially useful are electrochemical methods for detection of labeled particles within the membrane. Typically the particles will contain an enzyme or other catalyst which converts a substrate into products which can be detected electrochemically. The electrochemical methods included most generally are amperometric and potentiometric methods. An especially useful detection device is a photosensitive semiconductive electrode which responds to a detectable reaction product. This detection device has been described in U.S. 4,591 ,550 and by Hafeman et. al. (Science, vol.240, pp. 1182-1 185, 1988). The Threshold™ system (Molecular Devices, Menlo Park, CA) utilizes this device with a silicon oxynitride surface in contact with the solution phase which causes the sensor to respond to changes in pH. Alternately, a thin layer of gold may be deposited on the surface of the sensor which causes the sensor to respond to changes in the redox potential of an electrochemically active species in solution. When used with a pH sensitive electrode, the label will normally be an enzyme or catalyst which causes a change in pH, such as the hydrolysis of urea by urease. When used with a redox sensitive electrode, the label normally will be an enzyme or catalyst which causes a change in the redox potential, such as the hydrolysis of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase. The asymmetric membrane with captured particles containing detectable label will normally be saturated with other solution components required for signal generation, and can be compressed between a plunger on one side and the photosensitive semiconductor electrode on the other. In this manner, the detectable products are contained within a volume defined by the thickness and pore size of the compressed membrane. Other electrochemical detection devices could be utilized with equal advantage.

The microparticles containing bound specific binding pair member are useful for determining a wide variety of analytes in liquid samples. For example, to perform a one step sandwich immunoassay, sample containing analyte, is mixed with microparticles which contain an antibody which is specific for the analyte and a second antibody which also is specific for the analyte and which has a detectable label directly or indirectly attached to it. The microparticles, with associated analyte and antibodies, are captured within the membrane. After washing away unbound labeled antibody, the bound label associated with the microparticles is determined by and is related to the concentration of analyte initially contained in the sample.

For a two step sandwich immunoassay, sample containing the analyte of interest, is incubated with microparticles which contain an antibody complementary to the analyte. The microparticles with bound analyte are captured within the membrane. The analyte associated with the particles is then labeled by incubating with a second labeled antibody. After washing away unbound labeled antibody, the bound label associated with the microparticles is determined and is related to the concentration of analyte initially contained in the sample.

For a one step competitive immunoassay, microparticles containing bound antibody are incubated with sample containing the analyte of interest and an analyte or analyte analog containing a detectable label. The antibody will be at a limiting concentration with respect to the total analyte present. The microparticles, with associated antibody, analyte, and labeled analyte, are captured within the membrane. After washing away free labeled analyte, the bound label associated with the microparticles is determined by and is related to the concentration of analyte initially contained in the sample. In an alternate configuration of the one step competitive assay, microparticles contain bound analyte or analyte analog, and a limiting concentration of labeled antibody is in the solution phase.

For a two step competitive immunoassay, microparticles containing bound antibody are incubated with sample containing the analyte of interest. The microparticles with associated antibody and bound analyte are captured within the membrane. The particles are then reacted with the labeled analyte or analyte analog. The label binds to antibody sites which do not already contain free antigen. After washing away unbound labeled analyte or analyte analog, the bound label associated with the microparticles is determined and is related to the concentration of analyte initially contained in the sample. In a variation of this method, microparticles with associated antibody are first reacted with labeled analyte or analyte analog, and are reacted with sample after capture of particles within the membrane. In an alternate configuration, microparticles contain bound analyte or analyte analog, and a limiting concentration of labeled antibody is in the solution phase.

With the exception of the membrane capture and subsequent detection, the above description represents commonly performed immunoassay protocols and are in no way meant to be restrictive. For example, although the above examples refer to interactions of antibodies with their respective haptens and antigens, any specific binding pair members may be used. These other specific binding pair members would include biological receptor proteins and their respective ligands as well as complementary strands of nucleic acid sequences. In certain cases, analyte may be captured on the microparticles by non-specific means and reacted with a labeled specific binding pair member as above. Furthermore, the labeled specific binding pair member used in the above examples may be replaced with a different specific binding pair member which is in turn recognized by another labeled specific binding pair member. For example, a labeled specific binding pair member may be replaced by a specific binding pair member which contains a ligand which in turn is recognized by a specific binding pair member specific for said ligand which contains a detectable label. There are a wide variety of specific binding pair member interactions which can be used to measure substances of interest which will be evident to anyone skilled in the art.

Example I

This example demonstrates the improved capture and retention of microparticles on asymmetric membranes as compared with conventional membranes

A. REAGENTS

1 . Buffer A: 0.01 M NaPi, 0.1 M NaCI, 1 mM EDTA, 0.25% Triton X-100, pH 6.5.

2. ^{1 25}l labeled anti-(β)hCG (mouse, monoclonal) coated polystyrene microparticles. The particles were identical to those used in the IMx® B-hCG reagent pack (No. 3A63-20) (Abbott Laboratories, North Chicago, IL. 60064). Microparticles were iodinated according to the Bolton-Hunter method (Biochemical Journal 133, 529-539, 1973). 10 μL of 0.1 mg/mL N-succinyl 3-(4-hydroxyphenyl)propionate (NSHPP) in ethanol was evaporated to dryness in the bottom of a glass test tube. The residue was dissolved in 0.1 mL of 0.5M sodium phosphate (pH 7.5). Na^{1 25}l was added (4.0 mCi in 21 μL) followed by 50 μL of chloramine-T (3.5 mg/mL in distilled water). The reaction was quenched by adding 55 μL of sodium metabisulfite (3.5 mg/mL in distilled water). The reaction mixture was extracted sequentially with 300 μL benzene/10 μL dimethylformamide followed by 300 μL benzene. The extracts were combined, dried, and the residue dissolved in 0.1 M borate buffer (pH 8.5). 1 mL of antibody coated microparticles (1 % solids, washed and resuspended in 0.1 M sodium borate (pH 8.5)) was added and the reaction was allowed to proceed for 30 minutes at room temperature. The reaction was stopped by adding 0.5 mL of 0.2M glycine buffer (pH 8.0). The particles were sedimented (3 min in Eppendorf™ centrifuge at 4°C), and resuspended in 0.05M Tris, 0.1 M NaCI, 13.6% sucrose, 1% BSA, 0.1% NaN3. The particles were washed by centrifugation and resuspended in the above buffer until the background counts in the supernatant were less than 2x background. The final suspension contained 0.5% solids with a specific activity of 78,000 cpm/μg solids. The labeled particles were further diluted by mixing 10 μL of this suspension with 5 mL of unlabeled anti-(β)hTSH (mouse, monoclonal) coated polystyrene microparticles and 50 mL of buffer A containing 1 mg/mL bovine serum albumin. The resulting suspension contained 100 ug/mL of particles and a specific activity of 800 cpm/μg particles. Each day prior to use, the particles were pelleted by centrifugation for 5 minutes at 1500xg, resuspended in buffer A containing 1 mg/mL bovine serum albumin, and sonicated for 15 seconds in ε Model B-2200R-1 Bransonic™ ultrasonic cleaner (Branson Cleaning Equipment Company, Shelton, CT).

B. MEMBRANES

The following membranes were used; important characteristics of these membranes as currently understood are summarized below. Detailed information concerning manufacturing processes for these membranes are proprietary and are not available.

1 . Filterite BTS 30 asymmetric polysulfone membrane (Memtek America Corporation, DeLand, FL.32724-9990). This is an asymmetric membranes whose pore size decreases from an average of 45μ on the upstream side to 0.45 μM on the downstream side.

2. Gelman Ultrabind SV-450. 0.45μ pore size 7 mil thick polysulfone membrane (Gelman Sciences, Ann Arbor, Mi., 48106) on a non-woven polypropylene support containing with active groups for covalently binding proteins. The active sites on the membranes were blocked prior to use by soaking the membrane in 10 mM adipic dihydrazide for 1 hour at room temperature. The membrane was then air dried at room temperature.

3. Gelman TR800. 0.8 μ pore size 7.5 mil thick hydrophilic polysulfone membrane (Gelman Sciences, Ann Arbor, Mi., 48106) on a non-woven polyester fabric support. 4. Gelman Ultrabind TR800. 0.8μ pore size polysulfone membrane (Gelman Sciences, Ann Arbor, Mi., 48106) on a fabric support containing active groups for covalently binding proteins. The active sites on the membranes were blocked prior to use by soaking the membrane in 10 mM adipic dihydrazide for 1 hour at room temperature. The membrane was then air dried at room temperature.

5. Gelman PSP450. 0.45μ pore size polysulfone membrane (Gelman Sciences, Ann Arbor, Mi., 48106) on a non- woven polypropylene support. 6. Gelman Versapor-450T: 0.45μ pore size acrylic copolymer on a nylon support(Gelman Sciences, Ann Arbor, Mi., 48106).

7. Gelman AP-450: 0.45μ pore size acrylic copolymer on a polyester support(Gelman Sciences, Ann Arbor, Mi., 481 06) .

8. Gelman Supor 450: 0.45μ hydrophilic polysulfone membrane, unsupported(Gelman Sciences, Ann Arbor, Mi., 48106) .

9. Gelman Supor 800: 0.8μ hydrophilic polysulfone membrane (Gelman Sciences, Ann Arbor, Mi., 48106), unsupported.

10. Gelman HT Tuffryn-450: 0.45μ polysulfone membrane (Gelman Sciences, Ann Arbor, Mi., 48106), unsupported. 1 1 . Amicon Autoblock AB: 0.45μ membrane (Amicon

Division, W.R. Grace and Co., Beverly, Mass., 01915) designed for protein immobilization.

12. Amicon Autoblock AB: 1.2μ membrane (Amicon Division, W.R. Grace and Co., Beverly, Mass., 01915) designed for protein immobilization.

13. Pall Loprodyne: 0.45μ hydrophilic nylon 66 membrane (Pall Biosupport Co., Glen Cove, N.Y., 11582). C. MEASUREMENT OF MICROPARTICLE CAPTURE AND RETENTION

1 . Each of the above membranes was mounted onto the filter element utilized by the Threshold™ system manufactured by Molecular Devices, Inc. (Menlo Park, CA ). This filter element has a means for alignment within a filter assembly which allows solutions to be drawn through the membrane at a plurality of locations and also has a means for alignment in a reading assembly which allows these above locations to aligned with detection sites within a sensor assembly. The Threshold work station is described in detail in WO 90/08313. The membranes were mounted on the filter elements (threshold sticks) using double sided adhesive tape. Where asymmetric supported membranes were used, the membrane was mounted in the filter element such that the particles would enter the membrane on the more porous side (larger pore size) and be captured in the depth of the membrane. Orientation of the other membranes was not as critical. The filter elements with attached membranes were inserted between upper and lower portions of the filter assembly. The upper portion of the assembly contains wells into which solutions can be pipetted. The wells become progressively more narrow and at the base of the upper unit measure 2.7 mM in diameter. The base of the filter unit contain holes of the same diameter which align with those in the upper portion. The assembly was then clamped onto the vacuum manifold of the Threshold workstation. This work station allows solutions applied to the wells in the upper portion of the filter assembly to be drawn through the membrane mounted on the filter element into the vacuum manifold under specified vacuum levels. As a result of this operation, microparticles are retained at separate discrete locations on the membrane. In these experiments, the upper portion of the filter assembly was modified by drilling a 5/32" hole through the center. By pipetting buffer into this hole, the membrane adjacent to the capture position is effectively bathed in buffer which aids in reducing non-specific background signal due to small amounts of labeled tracer which diffuse in the plane of the membrane. This modification was useful in reducing background signal, but was not critical for the invention. 2. Capture positions were prewashed with 200 μL of buffer A containing 1 mg/mL bovine serum albumin under a vacuum of 1.5 inches Hg. Approximately 500 μL of buffer A containing 1 mg/mL of bovine serum albumin was pipetted into the drilled out hole in the top on the filter assembly. 100 μL of diluted microparticle suspension was added to each of the filter wells and the solution drawn through the membrane under a vacuum of 1.5 inches Hg. The captured microparticles were washed by adding 500 μL of buffer A to each of the wells and drawing the solution through the membrane under a vacuum of 7.5 inches Hg.

3. The filter element with attached membrane was removed from ^'the filter assembly and the membrane from two capture positions was punched from the membrane. The filter element with attached membrane was then inserted into buffer A and gently agitated for approximately 1 minute. The filter element with attached membrane was then removed from buffer A and membrane from two additional capture positions was punched from the membrane. The membrane was then inserted into the sensor unit containing buffer A. The membrane was compressed in the sensor unit between the plunger and photoresponsive electrode and subsequently released. In this manner, the membrane was subjected to the same treatment as would occur during a read cycle. The filter element was then removed from the sensor unit and the membrane from the 4 remaining capture positions was punched from the membrane.

4. The total radioactivity associated with each of the membrane samples was determined. From the known amount of radioactivity initially applied, the percent of applied particles remaining on the membrane at each stage in the above sequence of treatments was determined.

The above results demonstrate the improved retention of microparticles by asymmetric and supported membranes. An exception was with Versapor-450T and AP-450 membranes. Both of these membranes are supported acrylic copolymers and the difference in porosity between the two faces of the membrane was not as evident visually as with the other asymmetric and supported membranes.

Example II

This example describes a one step sandwich immunoassay for hTSH is accordance with the current invention. It further demonstrates the improved retention of microparticles captured at the interface of supported laminated membranes of dissimilar pore size as compared to a membrane of single pore size. A. REAGENTS

1 . Anti-(β)hTSH (mouse, monoclonal) coated polystyrene microparticles in buffer with protein stabilizers. The particles were identical to those used in the IMx® Ultrasensitive hTSH Reagent Pack (No. 3A62-20) (Abbott Laboratories, North Chicago, II. 60064). Particles were diluted 1 :1 in 0.05M Tris, 0.1 M NaCI, 13.6% sucrose, 1% bovine serum albumin, pH 8.0 prior to use. The final particle concentration was 0.05%.

2. Anti-(a)hTSH (mouse, monoclonal) antibody-urease conjugate. The conjugate was prepared by reacting the antibody with a 5 fold molar excess of succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) in 0.05M NaPj, 0.15M NaCI, 1 mM EDTA, pH 7.0 as described in Olson et al. (J. of Immunological Methods, 134(1 ) 71-79 (1990)). The derivatized antibody was mixed with urease in this same buffer and coupling was allowed to proceed for 3 hours at room temperature. The conjugate was purified by gel filtration on a Sephacryl S-400 column equilibrated with 0.01 M NaPj, 0.15M NaCI, 1 mM EDTA, 0.1 M Na2SO4,0.01% NaN3, pH 7.0. The conjugate was diluted in 0.1 M Tris, 0.5M NaCI, 1 mM EDTA, 2% fish gelatin, 0.5% non-fat dry milk, 1% brij-35, 0.1% sodium azide.pH 7.2 and was used at a final concentration of 6.2 μg/mL.

3. Buffer A: 0.05M Tris, 0.3M NaCI, 0.1% sodium azide, pH 7.5.

4. Buffer B: 0.01 M NaPi, 0.1 M NaCI, 1 mM EDTA, 0.25% Triton X-100, pH 6.5 5. Substrate buffer: 0.1 M urea in wash buffer B.

6. IMx® Ultrasensitive hTSH Calibrators (No. 3A62- 01 ): hTSH in porcine serum at concentrations of 0, 0.5, 2.0, 10.0, 40.0, and 100.0 μlU/mL (Abbott Laboratories, North Chicago, II. 60064).

B. MEMBRANES

1 . Nitrocellulose. Biotin-BSA coated nitrocellulose membrane (0.45μ) purchased from Molecular Devices, Inc. (Menlo Park, CA 94025).

2. Nylon. Cuno Zetapor 45SP membrane (0.45μ, 6.5 mil. Nylon 66 membrane) 3. Supported polysulfone. Gelman Ultrabind SV-450 membrane. Activated 0.45μ, 7 mil polysulfone membrane on a fabric support. This membrane contains active sites which react with amino groups on proteins. These active sites on the membrane were not blocked prior to use.

C. ASSAY FOR hTSH.

1 . 150μL of sample calibrator containing from 0-100 μlU/mL of hTSH was mixed with 150 μL of buffer A, 150 μL of microparticle reagent, and 150 μl antibody-urease conjugate and incubated for 30 minutes at 37° C .

2. Each of the above membranes was mounted onto the filter element utilized by the Threshold system manufactured by Molecular Devices, Inc. (Menlo Park, CA ). The membranes were mounted on the filter elements (threshold sticks) using double sided adhesive tape. The Gelman membrane (ultrabind SV-450) was mounted on the filter element such that the fabric backing would be adjacent to the applied reaction mixture. Orientation of the other membranes was not critical. The filter elements with attached membranes were inserted between upper and lower portions of the filter assembly. The assembly was then clamped onto the vacuum manifold of the Threshold workstation. 3. 175 μl of the above reaction mixtures were drawn through each of the above three membranes at a vacuum level of approximately 1.5 inches Hg. After all of the reaction mixtures had been drawn through the membrane the immobilized microparticles containing bound immunocomplexes were washed two times sequentially with 175μL of buffer B. The buffer B was drawn through the membrane at a vacuum level of 7.5 mM Hg.

4. Upon completion of the second wash, each of the filter element with associated membranes was removed from the filter assembly and placed in buffer B for 1 minute. Each of the filter elements was then placed into the reader assembly for measurement of enzyme activity. After the activity of enzyme associated with each of the filtration positions on each element had been determined, the element with associated membrane was stored in buffer B. After all membranes had been read once, membranes were reinserted into the reader assembly and read sequentially an additional 2-5 times. Membranes were stored in buffer B between subsequent reads.

The enzyme activity associated with the immobilized immunocomplexes was measured using a light addressable potentiometric sensor (LAPS) in a Threshold™ work station. The sensor is described in US 4,591 ,550. It is a photoresponsive insulated semiconductor device which responds to surface potentials at the electrolyte-solid interface. The potential can be measured at multiple sites essentially simultaneously by illuminating the chip at multiple sites using an intensity modulated light source. The reader assembly is described in WO 90/08313. The reader assembly contains the photoresponsive electrode, and a plunger, both immersed in substrate buffer. Included in the assembly is a means for alignment of the filter element within the assembly and a means for compressing the membrane portion of the filter element between the plunger and the photoresponsive electrode.

In this particular experiment, the sensor was coated with a silicon oxynitride coating which causes the sensor to respond to changes in pH. Urease activity is measured by the increase in pH caused by hydrolysis of urea.

D. RESULTS

The results are shown in the table below.

Rate (μV/sec. L

For the first read in the above experiment, membranes were removed from the filter assembly, placed in buffer B for 1 minute, and then inserted into the reader unit. The second read was performed after the membrane had been soaked in buffer B for 10 minutes. Read 3 was performed after soaking for an additional 10 minutes. Read 4 was performed after the membrane had been soaked in buffer B for a total of 1 hour. Read 5 was performed after the membrane had been soaked overnight in buffer B. Buffer B was kept at room temperature. In this experiment, microparticle retention is best estimated from the enzyme activity of the highest sample (100 μlU/mL). Loss of enzyme activity at lower hTSH levels is influenced to a greater extent by general reduction in non¬ specific binding from read to read and dissociation of antibody-urease conjugate from the immobilized microparticles. The 100 μlU/mL samples on both the nitrocellulose and nylon membranes lost 85-90% of their initial activity between the first and second read. In contrast, the supported polysulfone membrane lost less than 10% of the initial activity even after 4 reads for this sample. After standing overnight in buffer B, only 29% of the initial activity was lost, part of which may have been the result of enzyme inactivation .

Example III

This assay describes a two step sandwich immunoassay for hTSH in accordance with the current invention. In this experiment, active sites on the supported polysulfone membrane were inactivated prior to use. This lowered the assay background due to reduction of non-specific adsorption of enzyme conjugate and further demonstrated that the active sites were not required for capture and retention of particles.

A. REAGENTS.

1 . Anti-(β)hTSH (mouse monoclonal) coated polystyrene microparticles were the same as those used in Example II above. Particles were diluted 1 :5 in buffer B prior to use. The final particle concentration was 0.02%.

2. Anti-(a)hTSH(mouse, monoclonal) antibody urease conjugate was the same as that described in Example II. The conjugate was diluted in 0.1 M Tris, 0.5M NaCI, 1 mM EDTA, 2% fish gelatin, 0.5% non-fat dry milk, 1% brij-35, 0.1% sodium azide, pH 7.2 and was used at a final concentration of 10 μg/mL.

3. Buffer A, buffer B, substrate buffer, and hTSH calibrators were the same as described in Example II.

B. MEMBRANE

The membrane was a Gelman Ultrabind SV-450 supported polysulfone membrane containing active sites for protein immobilization, identical to that described in Example II. The active sites on the membranes were blocked prior to use by soaking the membrane in 10 mM adipic dihydrazide for 1 hour at room temperature. The membrane was then air dried at room temperature. C. ASSAY FOR hTSH

1 . 50 μl of sample calibrator containing from 0 to 100 μlU/mL hTSH was mixed with 50 μL buffer A and 50 μL of microparticle reagent. The mixture was incubated 30 minutes at 37⁰C.

2. The filter element, filter assembly, and reader assembly were the same as in Example II. The membrane was attached to the filter element such that when assembled into the filter assembly the fabric support would be adjacent to the applied reaction mixture. The membrane was prewashed with 1 mg/mL bovine serum albumin in buffer A prior to use.

3. 125 μL of each of the reaction mixtures was transferred to the filter block. Reaction mixtures were drawn through the membrane under a vacuum of 1.5 inches Hg. Each of the capture positions was washed with 200 μL of buffer B. Buffer B was drawn through the membrane under a vacuum of 7.5 inches Hg.

4. 25 μL of antibody-urease conjugate was added to each of the capture positions. The conjugate was allowed to incubate above the membrane for 7.5 min at room temperature. During this incubation period the conjugate was observed to slowly soak into the membrane. A vacuum of 7.5 inches Hg was applied to the membrane and it was subsequently washed two times with 200 μL of buffer B.

5. The filter element was removed from the filter assembly. Enzyme activity was determined as described in Example II with the exception that the 1 minute presoak in buffer B was eliminated, and the element was inserted directly into the reader assembly. After the first read, the membrane was soaked for an additional 13 minutes in buffer B. The enzyme activity was then measured a second time.

D. RESULTS

The results are shown in the table below.

This example demonstrates that the reactive groups on the membrane were not required for retention of microparticles, and further demonstrates the utility of the method for performing two step immunoassays.

Example IV

This example demonstrates the increased signal observed with microparticle capture of antigen vs. capture of antigen using a biotin-streptavidin system.

A. REAGENTS

1 . Anti-(β)hTSH (mouse monoclonal) coated polystyrene microparticles were the same as those used in Example II above. The particles were diluted 1 :6 in buffer B prior to use. The final particle concentration was approximately 0.016%.

2. Anti-(β)hTSH (mouse, monoclonal) antibody- streptavidin conjugate. Antibody was conjugated to streptavidin as described by Olson et al. (J of Immunological Methods, 134(1 ) 71-79 (1990)). The antibody was reacted with a 10 fold molar excess of SMCC in 0.05M NaPi, 0.15M NaCI, 1 mM EDTA, pH 7.0 for 2 hours at room temperature. Streptavidin was reacted with a 7 fold molar excess of succinimidyl 3-

(pyridyldithio)propionate (SPDP) in this same buffer for 1 hour at room temperature. The derivatized streptavidin was treated with dithiothreitol to expose free sulfhydryl groups. The conjugate was prepared by incubating 12 mg of derivatized antibody with 12 mg of derivatized streptavidin. The conjugate was purified by affinity chromatography using diaminobiotin agarose and gel filtration on a Sephacryl S-100 column equilibrated with 0.05M Tris, 0.15M NaCI, 0.025 NaN3, pH 7.5. The conjugate was diluted to a final concentration of 2.0 μg/mL in 0.05M Tris, 0.1 M NaCI, 1 mM EDTA, 13.6% sucrose, 1.0% bovine serum albumin, 1.0 ug/mL streptavidin, 0.1% NaN3, pH 8.0

3. Anti-(a)hTSH(mouse, monoclonal) antibody urease conjugate was prepared as described in Example II. The conjugate was diluted in 0.1 M Tris, 0.5M NaCI, 1 mM EDTA, 2% fish gelatin, 0.5% non-fat dry milk, 1% brij-35, 0.1% sodium azide, pH 7.2 and was used at a final concentration of 10 μg/mL. The conjugate was prefiltered through a 0.2μ cellulose acetate filter prior to use. For experiments with biotin- streptavidin capture, the concentrations of fish gelatin and non-fat dry milk in the conjugate diluent were 8% and 2% respectively.

4. Buffer A, buffer B, substrate buffer, and hTSH calibrators were the same as described in Example II

B. MEMBRANES

1 . Supported polysulfone. Gelman Ultrabind SV-450, blocked with 10 mM adipic dihydrazide for 1 hour prior and air dried at room temperature as described in Example III. 2. Biotin-nitrocellulose. Biotin-BSA coated nitrocellulose membranes from Molecular Devices, Inc. (Menlo Park, CA 94025).

C. ASSAY OF hTSH USING MICROPARTICLE CAPTURE

1 . Sample calibrator containing from 0 to 100 μlU/mL hTSH were mixed with equal volumes of buffer A and microparticle reagent. The mixture was incubated for 30 minutes at 37°C . 2. The filter element, filter assembly, and reader assembly were the same as in Example II. The blocked polysulfone membrane was attached to the filter element such that when assembled into the filter assembly the fabric support would be adjacent to the applied reaction mixture. The membrane was prewashed with 1 mg/mL bovine serum albumin in buffer A prior to use 3. 150 μL of each of the reaction mixtures (containing a 50μL equivalent volume of serum) was transferred to the filter block. Reaction mixtures were drawn through the membrane under a vacuum of 1.5 in. Hg. Each of the capture positions was washed with 250 μL of buffer B. Buffer B was drawn through the membrane under a vacuum of 7.5 inches Hg.

4. 25 μL of antibody-urease conjugate was added to each of the capture positions. The conjugate was allowed to incubate above the membrane for 7.5 minutes at room temperature. A vacuum of 7.5 in. Hg was applied across the membrane. After remaining conjugate had been suctioned, the membrane was washed with 0.5 mL of buffer B.

5. The filter element was removed from the filter assembly. Enzyme activity was determined by inserting the stick into the reader assembly as described in Example III

D. ASSAY OF hTSH USING BIOTIN-STREPTAVIDIN CAPTURE

1 . 200 μL of sample calibrator containing from 0 to 100 μlU/mL of hTSH was mixed with 900 μL of antibody- streptavidin conjugate. The reaction mixture was incubated for 30 minutes at 37°C .

2. The filter element, filter assembly, and reader assembly were the same as in Example II. The upper portion of the filter assembly was drilled in the center as described in example I. The membrane was prewashed with 500μL of buffer B prior to use under a vacuum of 7.5 inches Hg.

3. 250 μL of each of the reaction mixtures (equivalent to 45 μL serum) was transferred to the filter assembly. Immunocomplexes were captured on the membrane via the biotin-streptavidin interaction by drawing the reaction through the membrane under a vacuum of 1.5 inches Hg. Each of the capture positions was washed with 500 μL of buffer B under a vacuum of 7.5 inches Hg.

4. Approximately 500 μL of buffer B was pipetted into the drilled out hole in the top of the filter assembly.

Antibody-urease conjugate (25 μL) was added to each of the filter positions and the conjugate was allowed to incubate above the membrane for 7.5 minutes at room temperature. Upon completion of the incubation, conjugate was drawn through the membrane under 1.5 inches Hg. The membrane was washed with 500 μL of buffer B under 7.5 inches Hg.

5. The filter element was removed from the filter assembly. Enzyme activity was determined by inserting the element into the reader assembly as described in Example III.

E RESULTS

Rate (μV/sec) rhTSHHi-il U/m L) Microparticle Capture Biotin-streptavidin capture

0 17.1 7.75

0.5 50.5 16.5

2.0 132 42.6

10 665 176

100 3236 3688

The hTSH standard curve slope between 0 and 0.5 μlU/mL obtained using microparticle capture was 3.8 fold greater than that obtained using biotin-streptavidin capture.

Example V

This example demonstrates a two step microparticle hTSH standard curve using alkaline phosphatase as enzyme label with potentiometric measurement of products using a gold coated photoresponsive semiconductive electrode sensitive to changes in redox potential. As can be seen from the results at the end of this example, the sensitivities of the redox assay in detecting minute quantities of analyte can be useful.

REAGENTS

1 . Anti-(β)hTSH (mouse, monoclonal) coated polystyrene microparticles in buffer with protein stabilizers. The particles were the same as used in Example II. The microparticles were diluted 1 :5 in 0.05M Tris, 0.1 M NaCI, 13.6% sucrose, 1% bovine serum albumin, 0.1% NaN3, pH 8.0. The final particle concentration was 0.02%. 2. Anti-(a)hTSH (goat) antibody-alkaline phosphatase conjugate. The conjugate reagent from the IMx® Ultrasensitive hTSH reagent pack (No. 3A62-20) (Abbott Laboratories, North Chicago, IL, 60064) was diluted 1 :10 in 0.1 M Tris, 0.5M NaCI, 2% fish gelatin, 0.5% non-fat dry milk, 1% brij-35, 0.1% NaN3, 1 mM MgCl2, 0.1 mM ZnCl2, pH 7.2 prior to use.

3. Buffer A: 0.05M Tris, 0.3M NaCI, 0.1% NaN3, pH 7.5.

4. Buffer B: 0.2M Tris, 0.2M NaCI, 1 mM magnesium acetate, 0.05% NaN3, pH 10.3 5. Buffer C: Buffer B containing 0.75 mM FeCl3, 0.25 mM FeCl2, and 3.3 mM o-phenanthroline

6. Substrate buffer: Buffer B containing 0.75 mM

FeCl3, 0.25 mM FeCl2, 3.3 mM o-phenanthroline, and 5 mM 5- bromo-4-chloro-3-indolyl phosphate (BCIP). 7. hTSH calibrators were the same as Example II.

B. MEMBRANE

1 . Filterite BTS30 asymmetric polysulfone membrane (Memtek America Corp., Deland, FL 32724-9990).

C. ASSAY FOR hTSH

1 . 25 μL of sample calibrator containing from 0-40 μlU/mL of hTSH was mixed with 75 μL of buffer A and 50 μL of diluted microparticle reagent. The mixture was incubated for 30 minutes at room temperature.

2. The filter element, filter assembly, and reader assembly were the same as in Example II. The upper portion of the filter assembly was drilled in the center as described in Example I. The membrane was attached to the filter element such that when assembled into the filter assembly the more porous side of the membrane would be adjacent to the applied reaction mixture. Each position on the membrane was prewashed with 500 μL of buffer B prior to use under a vacuum of 7.5 inches Hg.

3. 125 μL of each of the reaction mixtures was transferred to the filter block. Reaction mixtures were drawn through the membrane under a vacuum of 1.5 inches Hg. Each of the capture positions was washed three times sequentially with 200 μL of buffer A. Buffer A was drawn through the membrane under a vacuum of 7.5 inches Hg. 4. Approximately 500 μL of buffer B was pipetted into the drilled out hole in the top of the filter assembly. 25 μL of diluted antibody-alkaline phosphatase conjugate was added to each of the capture positions. The conjugate was allowed to incubate above the membrane for 7.5 min at room temperature. During this incubation period the conjugate was observed to slowly soak into the membrane. A vacuum of 1.5 inches Hg was applied to the membrane for 1 minute. It was then washed sequentially with (2x) 200 μL of buffer A and (2x) 200 μL of buffer B under a vacuum of 7.5 inches Hg.

5. The filter element was removed from the filter assembly. The membrane was allowed to soak in buffer C for 1 minute. Enzyme activity was then determined by inserting the stick into the reader assembly as described in Example II. In this example, the sensor was coated with a thin layer of gold which caused the sensor to respond to changes in redox potential caused by alkaline phosphatase hydrolysis of BCIP.

The results shown here indicate the effectiveness of using the present invention and monitoring redox potential changes. The sensitivity of this assay can be useful in detecting small levels of analyte in test samples.

Claims

We claim:

1 ) A method for determining the presence or amount of an analyte in a test sample, comprising: forming a test mixture by contacting the test sample sequentially or simultaneously with an indicator reagent and a capture reagent, said indicator reagent comprising a detectable label directly or indirectly attached to a first specific binding pair member, said capture reagent comprising a second specific binding pair member directly or indirectly attached to a microparticle, allowing the analyte in said test sample to bind to at least said capture reagent; immobilizing said microparticles by contacting said test mixture with an asymmetric membrane wherein one side of said membrane has pores of a size sufficient to allow said microparticles to enter and a second side of said membrane has pores of a size prohibiting the passage of said microparticles therethrough; placing said membrane in an inverted or vertical position; monitoring said detectable label within said membrane to determine the presence or amount of said analyte in said test sample.

2) The method of claim 1 further comprising submerging said membrane in an aqueous solution.

3 ) The method of claim 1 wherein said microparticles immobilization occurs prior to forming the test mixture.

4) The method of claim 1 wherein said microparticle immobilization comprises drawing said microparticles into the said membrane by capillary flow.

5) The method of claim 1 whereby the said microparticle immobilization further comprises drawing said microparticles into said membrane by the application of a pressure differential to opposite sides of said membrane.

6) The method of claim 5 wherein said pressure differential is a vacuum. 7) The method of claim 1 wherein said asymmetric membrane comprises a decreasing pore size gradient.

8) The method of claim 7 wherein said decreasing pore size gradient is substantially continuous from one side of said membrane through to opposite side of said membrane.

9) The method of claim 7 wherein said asymmetric membrane has a decreasing pore size gradient from the larger pore size on one side of the membrane to the center of the membrane, then an increasing pore size gradient from the center of the membrane to the other side of the membrane.

10) The method of claim 1 wherein said asymmetric membrane comprises a microporous membrane and a support wherein said microporous membrane has a pore size sufficient to prohibit said microparticles from passing therethrough and said support has a pore size sufficient to allow the passage of said microparticles into said asymmetric membrane.

1 1 ). The method of claim 9 wherein said microparticles are immobilized at or about the interface of said microporous membrane and said support.

12) The method of claim 1 wherein said detectable label reacts with or is catalyzed by a substrate to produce a detectable signal.

13). The method of claim 2 wherein said aqueous solution comprises a substrate for the detectable label.

14) The method of claim 1 whereby said monitoring comprises detecting said detectable label from the interior of said asymmetric membrane.

1 5) The method of claim 14 whereby said detecting comprises measuring pH or pH change.

16). The method of claim 14 wherein said detection comprises the measurement of the redox potential change. 1 7) . The method of claim 14 wherein said detectable label is a flurophore.

1 8) The method of claim 1 wherein said monitoring occurs by subjecting said asymmetric membrane to a physical force to affect a physical contact between said asymmetric membrane and a detector or sensor.

1 9) The method of claim 1 wherein said asymmetric membrane is held in an inverted or vertical position during said monitoring.

20) The method of claim 1 wherein said analyte is thyroid stimulating hormone.