WO1990001167A1

WO1990001167A1 - Porous support system for the immobilization of immunoassay components and assays performed therewith

Info

Publication number: WO1990001167A1
Application number: PCT/US1989/002204
Authority: WO
Inventors: Marina Adamich; Alan Robert Craig; Alice Ann Jarvis; Charles Clayton Leflar; Harry W. Meslar; Robert Philip Saltman; Donald Max Simons; Chi-Chin Wang
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1988-07-19
Filing date: 1989-05-25
Publication date: 1990-02-08

Abstract

This invention relates to the immobilization of reagents to a fused porous polymer support and the use of this support in performing heterogeneous assays of components of human and animal fluids and tissues. In particular, this immobilization refers to covalent reactions of the solid support to reagents where these reagents include but are not limited to enzymes, antibodies, and antigens. Novel and facile methods for effecting protein attachment to the support are disclosed.

Description

TIT E

POROUS SUPPORT SYSTEM FOR THE IMMOBILIZATION OF

IMMUNOASSAY COMPONENTS AND ASSAYS PERFORMED THEREWITH

BACKGROUND OF THE INVENTION Immunoassays are common tools used in the analysis and quantisation of the components of tissues such as plasma, serum, blood, urine, cerebrospinal fluid, and amniotic fluid. These assays generally require the immobilization of some component of the assay such as an antibody, an enzyme, or an antigen, and a set of non- immobilized soluble components such as antibodies, antibody with markers (i.e., enzymes, radioisotopes) attached, analytes and analytes with markers attached. The successful completion of an assay requires: the intimate contact of the immobilized components, the soluble components, and the sample; sufficient time of incubation for the interaction of the components usually including the binding of soluble components to the immobilized components; and the separation of the remaining soluble components from the solid support. Detection can be effected by measuring the amount of soluble component remaining or by measuring the amount of previously soluble component which is now bound to the solid, immobilized phase. A number of methods for immobilizing assay components have been disclosed in the literature. These methods can be divided into three general categories; physical entrapment, adsorption, and covalent attachment. Of the three, covalent attachment is preferred. Entrapment has the disadvantage of maintaining physical inaccessibil¬ ity of the portions of the immobilized components which must contact soluble components. Supports designed to immobilize by adsorption frequently exhibit the undesirable non-specific adsorption of other components during the assay. Adsorption and entrapment share the serious disadvantage of being impermanent and changes in temperature, ionic strength, pH, buffer components, physical agitation, and many other parameters may cause the release of previously bound components.

A more satisfactory method for forming the immobilized component is to create a covalent chemical bond between this component and the surface of the solid support. Many comprehensive surveys of covalent immobilization techniques have been published; for example, methods in Enzymology, Volume XLIV: Immobilized Enzymes, Academic Press, 1976; Immobilized Enzymes for Industrial Reactors, Academic Press, 1975; Review of Pure and Applied Chemistry, Vol. 21, #83: Insolubized Enzymes, Biochemical Applications of

Synthetic Polymers. Many of these coupling reactions suffer from the disadvantage of necessitating the use of chemicals (i.e., carbodi- imide, glutaraldehyde) which could cross-link the component of interest, forming aggregates which exhibit sub-optimal performance in the assay and which may not be successfully immobilized. The most successful immobilization techniques frequently involve first the introduction or generation of reactive groups on the support surface, removal of soluble reactive material, followed by the addition of the component of interest. This has the advantage of avoiding cross- linking of material but may suffer from several disadvantages such as an insufficient density of reactive sites, reactive groups may be hydrolized or deactivated prior to the addition of the component to be immobilized, and the support itself may be made less desirable by the activation process. Physical and chemical manipulation can weaken or destroy gels and polymers and create undesirable pore- clogging or fines which prevent effective contact and separation at latter stages of the assay.

A wide choice of solid supports for immobilizations are available and the above listed reviews discuss many of them. Supports based on polymerized biological materials such as dextran, cellulose and agarose do provide high surface area but are subject to chemical, biological and physical degradation and often exhibit poor flow characteristics especially with high pressure or high flow rates. Inorganic supports such as silica gel or alumina are unsatisfactory in that it is difficult to maintain the necessary open pore diameter and pore volume for high flow rates when a column or filter will be used to effect separations and it may be difficult to attach reactive groups to these inorganic supports.

Good flow characteristics, physical, chemical, and biological stability and ease of separations can be achieved by use of a solid plastic polymer as the support (U.S. Patent #4,317,810 issued March 2, 1982, to S. P. Halbert and M. Auken). These supports require no packing within a column nor any centrifugation or filtration for their removal to effect separation of bound and unbound components in an assay. They exhibit excellent mechanical stability and do not support the growth of microorganisms. The polymer support base can be stored dry and requires no rehydration time. *'

Solid, synthetic polymer supports have major drawbacks. A small surface area is available - compared to supports consisting of dextran, agarose or cellulose, etc. - and the surfaces are genera ly only suited for adsorption, not covalent attachment. The practical consequences of a small surface area are many. The amount of the immobilized component which can be attached is severely reduced, and the surface area to volume ratio within the assay is so low that soluble components must diffuse some distance to encounter the surface components. This increases the length of time required in all incubation steps and, therefore, increases total assay time. These low-surface area solid polymer supports usually require agitation steps or continuous agitation during incubation periods to insure adequate contact with soluble components. These drawbacks combine to make assays using these supports inefficient, time consuming and labor intensive.

SUMMARY OF THE INVENTION The present invention is directed to the use of a porous solid support system for the immobilization of a component or components of an assay. In a preferred embodiment, the solid support is a single piece of plastic manufactured by fusing or sintering or partially solvating a finely ground mixture of solid polymer resin to produce an open pore structure through which liquids can flow, contacting the entire surface area. Such a material is available commercially (Porex Technologies, Inc., Fairburn, GA; Cromex Corp., Brooklyn, NY; General Polymeric Corp., West Reading, PA) and is most frequently used for producing pen tips. Unique advantages result from the use of a support with pore sizes in the 1 to 500 micron range for performing assays on biological materials. Facile methods for immobilizing assay components are disclosed, involving the deposit of a thin layer of reactive polymer on the large surface area presented by the porous plastic support, followed by reaction with the component to be immobilized. Assays for digoxin, TSH, hCG, and fern^"tin in human sera are described. DETAILED DESCRIPTION OF THE INVENTION A porous plastic support for the immobilized component of an assay combines the well-known advantages of a particulate natural polymer gel (cross-linked dextran, cellulose, agarose) with those of a solid polymer support. It has a very high surface area to volume ratio, reduces incubation times to a minimum, permits liquids to thoroughly mix by virtue of the intricate network of connected pores, and allows excellent flow characteristics under high pressure and flow rates. Supports are not biodegradable, are mechanically stable, can be stored dry, require no rehydration time, and separation of bound and unbound assay components is achieved simply by drawing liquid through the support.

Porous plastic supports are commercially available and/or can be formed from a variety of polymer materials including polyethylene, polypropylene, ethylene/vinyl acetate copolymer, polyvinylidene fluoride, styrene/acrylonitrile copolymer, polytetra- fluoroethylene, polyamide and polystyrene. Supports may be formed from a single polymer or from a mixture of polymers or from a mixture of polymers and another particulate material such as charcoal, silicas, aluminas or ion exchange resins. In some cases, the desired separation of some components from the soluble phase may take place because of absorption on or reaction with the raw plastic support or due to interaction with the entrapped additive. The preferred embodiment is to covalently attach the immobilized component to the internal and external surfaces of the porous plastic support.

The shape and size of the support can vary widely and still achieve required assay performance. The preferred shape is a cylindrical column with a height exceeding the width. This overly elaborate explanation is equally true whatever the height (or thickness) of the support. The important factor is the residence time of the soluble component in the support matrix. At a given flow rate through the system, the residence time is independent of the geometry of the matrix provided that the volume of the support is constant. The size of the support should be balanced with the sample ; size, the amount and concentration of soluble component to be entrapped, the pore size - which determines the average distance a soluble component must diffuse to meet the immobilized component, the desirable incubation time on, or flow rate through the support, and the affinity of the immobilized component for the soluble component.

The pore size of the support affects the efficiency and rapidity of the binding of soluble components to the support. A wide range of pore sizes may be acceptable, but the preferred size is less than 500 microns. As pore size increases the advantages of high surface area to volume ratio and short diffusion distance decreases. Porous plastics with pore sizes under 20 microns are not available commercially so assays have been tested down to and including this practical pore size limit. Since assays have been successful at the 20 micron pore size and there is no trend for decreasing performance with decreasing pore size for some assays, no lower limit on acceptable pore size is expected so long as the pore size does not approach the molecular diameter of the soluble component.

The immobilized components of the instant invention are ligands or binder defined as follows. By ligand is meant an antigen, hapten, nucleic acid, enzyme substrate, vitamin, dye or other small organic molecule including enzyme substrates, effectors and inhibitors; and by binder is meant an antibody, enzyme, nucleic acid, binding protein, synthetic mimic of a- binding protein such as polylysine and polyethyleneimine or other biomacromolecule capable of specific binding in the manner of an enzyme/substrate, etc.

Attachment of the immobilized component to the plastic surface may be achieved with many conventional reagents well known in the art such as glutaraldehyde coupling or carbodiimide coupling to functional groups in plastics such as nylon. The preferred embodiment is to deposit a thin layer of an inherently reactive polymer over the internal and external surfaces of an otherwise inert porous plastic and follow this with exposure to the component to be immobilized. Successful assays using protein immobilization on chloromethylstyrene/styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide or a chlorosulfonated polyethylene such as the product Hypalon^®, available from E. I. du Pont de Nemours & Co., are all described below. Additionally, fabricated polystyrene objects may be treated with chlorosulfonic acid to introduce active sulfonyl chloride groups onto the surface. These active groups will readily react with free amino groups on -proteins to give sulfonamide bonds. Deposit of reactive polymer may be achieved by dissolving the polymer in solvent, wetting the porous plastic with this solution, and evaporating the solvent. Preferred solvents are highly volatile and have a composition such that they easily wet the porous plastic support without the use of detergents. These supports do not readily take up water-based solutions unless additives that reduce surface tension are preseϊht. The porous plastic support covered with a coating of reactive polymer is referred to herein as an activated support or activated bead.

Reaction of the component to be immobilized with the activated support requires complete contact of the internal and external surfaces of the activated support with the solution containing the component. These components may, therefore, be dissolved in liquids which are or which contain a percentage of a solvent with a low surface tension such as an alcohol. The activated support will then exhibit a sponge-like action toward the solution and total contact ts achievable at atmospheric pressures. In many desirable assay configurations, the immobilized component is a biologically active protein which might be adversely affected by small quantities of the required solvent. An alternate method of introducing liquid into the activated support is by the use of pressure changes. The two preferred embodiments are as follows: reduce the pressure of a vessel containing the activated support immersed in the component solution in order to reduce the quantity of air trapped in the support, followed by an increase of pressure to atmospheric or above to force the liquid to enter the pores replacing the air. This procedure may be repeated as necessary to achieve the desired penetration of liquid into the pores. An alternate embodiment is to evacuate air from a vessel containing only activated support and then release the resulting vacuum by the introduction into the vessel of a solution containing the component to be immobilized. The reaction with the activated support is conducted under conditions not considered harmful to the material to be immobilized. For proteins, this is generally a temperature between 0°C and 40°C. Other types of components may be more effectively immobilized at higher temperatures. A number of assay components have been successfully immobilized on the porous supports including antibodies, haptens attached to protein carriers, enzymes, and a binding protein, avidin. The present invention is not limited to the immobilization of substances in these categories. The following examples illustrate some of the uses of this invention but are not meant to cover the entire range of potential applications of these devices.

EXAMPLE 1 Preparation of Quabain-BSA Conjugates Ouabain octahydrate (5.0 g) was dissolved in 500 L of hot distilled water and allowed to cool to room temperature. Sodium metaperiodate (NaI04; 7.3 g) was added to the ouabain solution, and the mixture stirred for two hours in the dark. The solution was passed through a bed of Dowex (I-X8) anion exchange resin (prepared by washing 250 g of the Dowex resin with water until the yellow color disappeared). The oxidized ouabain solution was mixed with 500 mL of 1 M sodium phosphate (pH 7.0) containing 10 g of BSA. The mixture was stirred for one hour, 0.64 g of sodium cyanoborohydride (NaCNBH3) added, and stirring continued for 48 to 72 hours at room temperature. The resulting ouabain-BSA conjugate was dialyzed against running water for 12 to 24 hours and against 20 volumes of 0.015 M sodium phosphate buffer (pH 7.0) at 4°C for 16 hours. The conjugate- was stored at 4°C.

EXAMPLE 2 The following example relates to the use of the invention in performing assays for digoxin in human serum. a. Manufacture of Ouabain-BSA Conjugates See Example 1. b. Coating the Porous Plastic Support with Hypalon⁰ 40

The porous plastic supports used in this work were obtained from the Porex Corp. Shape U 11, in a range of porosities, was chosen for ease of manipulation and constant void volume over a range of pore sizes. Shape U 11 is a cylinder with one end flat and the other rounded. These objects are referred to as 'beads' in the following examples.

A batch of 200 Porex beads (pore size 20 μm) was placed in a glass beaker and completely submerged in 120 mL of a 0.1% solution of Hypalon^® 40 in dichloromethane. The system was subjected to a vacuum for approximately 10 seconds to evacuate air from the bead pores. Subsequent release of the vacuum efficiently filled the internal volume of the bead with the solution. Excess unabsorbed solution was decanted from the beads which were then dried in a vacuum oven at 45°C for two hours. The activated beads were now ready for attaching protein conjugate. c. Treating Activated Beads with Protein Solution

The activated beads were covered with approximately 120 mL of a solution of ouabain-BSA (0.83 mg/mL in 20% isopropanol/80% 12.5 mM phosphate, pH 7.0) and briefly subjected to a vacuum/pressure cycle to insure good filling of the internal volume. The assembly was stored in the dark overnight. d. Removal of Unbound Conjugate

Excess liquid was decanted from the treated beads, and the beads rinsed with 100 mL of 1% aqueous Triton X100, agitated on a rocker for two hours in 150 mL of fresh 1% Triton X100 solution, rinsed with 1% Triton X100 in isopropanol, and agitated in 250 mL of the 1% Triton X100 in isopropanol solution for two hours. Excess solution was decanted, and the beads dried overnight at 45°C in a vacuum oven. Beads were stored dry at 4°C until use. e. Assay for Digoxin in Serum

Assayed samples were filtered, normal human sera containing 0, 1, 2, 3, and 4 ng/mL digoxin. Each test sample was mixed with an equal volume of commercially available aca^® digoxin ABC reagent (E. I. du Pont de Nemours & Co., Wilmington, Delaware 19898) and allowed to react at room temperature for 10 to 30 minutes before use. Dried beads activated with ouabain-BSA (paragraph Id) were placed in bullet shaped cavities in an acrylic block, each cavity connected through tubing to a peristaltic pump. Exactly 60 μL of the test sample/aca digoxin ABC mixture were placed on top of each bead and this solution was pulled through the bead by the action of the peristaltic pump running at approximately 0.47 mL/min. This step was immediately followed by applying 200 μL of aca^® phosphate diluent to each bead. All eluents from each cavity were combined and collected in a test tube prior to assaying for eluted β-galactosidase activity. The assay was performed on a Cobas Bio (Roche Diagnostics) using 2-nitrophenyl galactoside (0NPG) in HEPES buffer as a substrate. f. Results of Assay for Digoxin in Sera

Data is displayed in terms of absorbance units at 404 nm.

Level # Replicates X abs. S.D. abs % CV

0 ng/mL 8 63.9 6.1 9.0 ^• 1 ng/mL 20 96.9 7.1 7.3

2 ng/mL 20 115.3 22.7 19.7

3 ng/mL 8 145.9 14.8 10.7

4 ng/mL 8 165.9 9.2 5.6

EXAMPLE 3 The following example describes the use of a 1:9 chloro- methylstyrene/styrene copolymer (prepared in-house by Dr. Alan Craig) for coating porous plastic beads with a reactive surface, and use of 5 the activated beads for assaying digoxin in human serum. a. Manufacture of Ouabain-BSA Conjugates See Example 1. b. Coating Porous Plastic with Chloromethylstyrene/styrene Copolymer

10 In preliminary experiments solutions of chloromethyl¬ styrene/styrene copolymer in dichloromethane ranging from 0.1% to 1% polymer were evaluated. A solution containing 0.5% polymer was used in this particular example. Porex beads (pore size 32 μm) were coated with polymer as in Example 2b. In this experiment, 100 beads

15 were prepared in one lot. c. Coating the Beads with Protein Solution

The reaction of the protein with chloromethylstyrene/ styrene copolymer-treated beads was carried out as in Example 2c with the exception that the solution contained 0.1 mg/mL ouabain-BSA. 20 d. Removal of Excess Protein Same as Example 2d. e. Assay for Digoxin in Serum

Same as Example 2c except assayed levels were 0, 1, 2, 3, 4, and 5 ng/mL digoxin. 25 f. Results of Assay for Digoxin in Sera

Assays were carried out on three successive days; all data are the mean of five replicates expressed in milliabsorbance units.

30

35

) EXAMPLE 4 The following example illustrates the flow rate dependence of the described procedure and establ shes the ideal flow range for this pore size. a. Preparation of Porous Plastic Beads

Activated Porex beads (32 μm pore size) were prepared as in Example 3a-e. b. Assay for Digoxin in Serum

Assays were carried out as in Example 2e, except calibrator levels were 0, 2.0 and 3.5 ng/mL and flow rates in the peristaltic pump were varied. Results were as follows; data is in milliabsorbance units per minute.

EXAMPLE 5 The following example describes the use of polybutadiene oxide in coating porous plastic with a reactive surface, and its use in performing digoxin assays on human serum samples. a. Preparation of Ouabain-BSA See Example 1. b. Coating of Porous Plastic with Polybutadiene Oxide

A 0.01% solution of polybutadiene oxide in dichloromethane was prepared. Twenty-four Porex beads (pore size 20 μm) as described in Example 1, were coated with this solution as described in Example 2, with the exception that volumes of solutions were reduced appropriately for the 24 bead lot size. c. Coating the Beads with Protein Solution

Protein coating with ouabain-BSA was carried out as in Example 2,c and 2,d with the exception that 12 beads were treated with 0.1 mg/mL ouabain-BSA and 12 beads with 1 mg/mL ouabain. d. Assay for Digoxin in Serum

Assays were carried out as in example 2,e with the exception that calibrator levels 0, 1, 2, 3, 4, and 5 ng/mL were tested. Results are as follows and are expressed in milliabsorbance units per in.

Calibrator 0.1 mg/mL Ouabain-BSA 1 mg/mL Ouabain-BSA 0 ng/mL 46.9 50.2

1 ng/mL 73.6 53.5

2 ng/mL 81.8 78.4

3 ng/mL 104.2 102.1

4 ng/mL 116.8 107.0 5 ng/mL 125.2 123.8

EXAMPLE 6 The following example describes the use of polyglycidyl methacrylate in coating porous plastic with a reactive surface, and its use in performing digoxin assays on human serum samples. a. Manufacture of Ouabain-BSA See Example 1. b. Coating the Beads with Polyglycidyl Methacrylate

A 10% solution of polyglycidyl methacrylate in 2-butanone was purchased from Polysciences, Inc. The working solution was made by diluting 1.0 mL of this stock with 19 mL of dichloromethane. About 25 beads were soaked in this solution, excess solution was decanted, and the beads dried at 45°C overnight. c. Coating the Beads with Protein Solution

Beads were coated with ouabain-BSA as in Example 2,c and 2,d, with the exception that the protein concentration was 16 mg/mL. d. Assay for Digoxin in Serum

Assays were carried out as in Example 2,e with the exception that calibrator levels were 0, 0.5, 1.25, 2.0, 2.75, and 3.5 ng/mL. Results are the mean of duplicates and are expressed as milliabsorbance units per min.

Calibrator Level X abs

.0 80.2 0.5 91.2

1.25 114.2

2.0 139.3

2.75 128.7

3.5 143

EXAMPLE 7 The following example describes a comparison of the relative performance of two reactive polymer coatings used on the porous plastic support. In order to avoid selecting factors such as a pore size or protein concentration that inadvertently favor one polymer coating over another, a co-optimization with a face centered cube statistical design was carried out with each polymer, a. Manufacture of Ouabain-BSA Conjugates See Example 1. b. Coating Porex with Reactive Polymer

This procedure was carried out as in Example 2,b for Hypalon^® coatings and Example 3,c for chloromethylstyrene/styrene copolymer coatings with the following exceptions. Pore sizes of the beads were 20, 38, and 51 microns, the polymer concentrations used in the coating solutions varied, as indicated below, between 0.01% and 1% polymer by weight. c. Coating the Beads with Protein Solution

The reaction of the protein with the activated beads was carried out as in Example 2,c with the exception that the concentration of the protein contact solution was varied between 0.01 and 1.0 mg/mL ouabain-BSA. d. Removal of Excess Protein, Depositing Detergent

Excess solution was decanted from the beads which were then placed, without rinsing, into a 45°C vacuum oven and dried overnight. The beads were then thoroughly wetted with a 1% Triton X-100 solution in propanol, excess propanol was decanted and the beads were again dried at 45°C. e. Assay for Digoxin in Serum

Assays were performed as in Example 2,e with the following exception. Calibrators with 0 and 4 ng/mL digoxin were run in triplicate. The results of the assay are expressed below in terms of the average milliabsorbance response at each level and the "Δ milli- A" between levels. Results indicate a superior performance level with Hypalon^® coatings as compared to chloromethylstyrene/styrene copolymer in coatings when the beads are prepared without wash steps. This level of performance indicates that the major portion of ouabain-BSA within the porous support was tightly bound to or reacted with the Hypalon^®. RESULTS WITH CHLOROMETHYLSTYRENE/STYRENE (CMS/S)

EXAMPLE 8 CHLOROSULFONATED POLYETHYLENE (HYPALON^® 40) ACTIVATION OF POREX FOR PROTEIN ATTACHMENT Porex (126-PE) beads were coated with a solution of 0.1% ^• Hypalon^® 40 in dichloromethane as follows: Porex beads were placed ^' in a beaker in a uniform, single layer and 0.1% Hypalon^® solution was added to thoroughly wet beads. Excess solution was removed and discarded, and the beads transferred to a clean beaker and vacuum- dried (45°C) for one hour. Dried, activated beads were stored at room temperature in the dark (foil wrapped) until use. Protein Attachment

Monoclonal, IgG anti 0.3 hCG antibody was purified from . ascites fluid by DEAE Sephadex column chromatography, dialyzed against phosphate buffered saline (PBS), lyophilized, and stored at -70°C. It was reconstituted by addition of sterile water. Antibody solution at a final calculated concentration of 10 μg antibody/bead^" was vacuum intruded to saturate porex micropores. The beads were post-blocked with PBS-0.1%. BSA excess blocking solution was removed by blotting and the beads lyophilized and stored at 4°C until use. Immunoassay Method

Calibrators, monoclonal IgG anti β-hCG-alkaline phosphatase Conjugate, 4-nitrophenyl phosphate (pNPP), wash and quench solutions for a sandwich β-hCG immunoassay were obtained from Hybritech TANDEM E-hCG kit reagents. Antibody-Porex beads were placed in individual holders in a plastic rack and pre-wetted with several milliliters of PBS-0.1% BSA solution. A 100 μL aliquot containing 50 μg calibrator (0, 25, 200 mlU/mL) and 50 μL antibody-enzyme conjugate was added to each bead and incubated 90 minutes at 37°C; this step was followed by three 2 to 3 L washes. Next, 100 μL of substrate pNPP was added to each bead. Substrate hydrolysis was allowed to proceed for 15 minutes at room temperature. The reaction was stopped by the addition of 1.0 mL quench reagent. Color development was then determined at 404 nm with samples blanked against quench reagent. The experiment was conducted in duplicate. Results Absorbance hCG (mlU/mL) at 405 nm

0 0.040

25 0.213

200 1.225

EXAMPLE 9 This example demonstrates the utility of the described invention for determining a multivalent antigen by a sandwich immunoassay technique, specifically the determination of ferritin in ? S human serum. a. Materials

The materials used in the example were rabbit polyclonal IgG antibody from Dako, Inc.; linear chloromethylstyrene/styrene copolymer (1:9) prepared in-house; dichloromethane from J. T. Baker, 10 Inc.; human serum albumin, buffer, salts, and Tween-20 from Sigma Chemicals Corp.; alkaline phosphatase-monoclonal antibody conjugate from Hybritech Corp.; porous plastic beads from Porex Corp.; and PEG- 8000 from Fisher Scientific Corp. b. Porex Selection

15 The U 11 shape was chosen for this experiment. Studies carried out with the same unit configuration but varying pore size demonstrated one optimal pore size range for ACMIA hapten applications (single site enzymetric immunoassays) and another for sandwich multivalent antigen assays (two-analyte enzymetric

20 immunoassays). The data are shown in Table 1. c. Solid Phase Reagent

A batch of 100 beads (pore size 170 μm) was immersed in 50 mL of 1% solution of chloromethylstyrene/styrene copolymer in dichloromethane for one minute. The excess solution was decanted,

25 and the beads were placed in a single layer on an aluminum tray, air dried for 10 minutes in a fume hood, and then placed in a vacuum oven at 45°C for 40 minutes. The activated beads were stored in a screw- capped polyethylene jar until use.

The activated beads were wetted with 70% aqueous

30 isopropanol to reduce surface tension, blotted dry on a paper towel, and then immersed in a solution of 50 Mg/mL of rabbit anti-human ferritin IgG in 100 mM HEPES buffer, pH 7.6 (50 mL of solution per 100 beads). This assembly was agitated for 24 hours at room temperature. The liquid was decanted from the beads and replaced

35 with 50 L of a solution of human serum albumin (1 mg/mL) in 100 mM HEPES buffer, pH 7.6. The assembly was agitated for 4 hours at room temperature and the solution decanted. The beads, now functionalized with rabbit anti-human ferritin IgG, were stored in a polyethylene screw-capped bottle at 4 to 8°C until used in the assay. d. Ferritin Assay Protocol

To 50 μL of serum was added 50 μL of monoclonal anti-human ferritin-alkaline phosphatase conjugate containing 4% PEG-8000 (weight/volume). This mixture was incubated at 37°C for 10 minutes. A bead functionalized with anti-ferritin IgG (from (c) , above) was added to the test solution, which wicked up into the porous structure. The bead was incubated at 37° for 10 minutes, then washed with 10 to 12 mL of wash buffer (150 mM NaCl, 0.2% Tween-20) by drawing the liquid through the bead with suction. The excess fluid was removed by suction, the bead placed in a clean 12 x 75 mm test tube, and 200 μL of substrate solution (10 mM PNPP in 0.5 M AMP buffer, pH 10.1) added. After incubation at 37°C for 30 minutes, 1.9 mL of quench buffer (150 M phosphate, pH 7.8) was added. The absorbance of each tube was then measured at 405 nm. The data are shown in Table 2.

10

Claims

What Is Claimed Is:

1. A porous, solid support for the immobilized component of an immunoassay, said support having an open pore structure through which liquid can flow, said component being attached to the porous solid support, said porous, solid support including a thin layer of a reactive polymer deposited thereon for receiving the component to be immobilized.

2. The porous, solid support of Claim 1 wherein the component is covalently attached to the reactive polymer on the porous, solid support.

3. The porous, solid support of Claim 2 wherein the reactive polymer is selected from the group comprising chloromethyl¬ styrene/styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.

4. A porous, solid support for the immobilized component of an immunoassay, said support having an open pore structure through which liquid can flow, said pores having a pore size of from about 2 to about 500 microns, the solid support being formed from a polymer selected from the group comprising polyethylene, polypropylene, ethylene/vinyl acetate copolymer, polyvinylidene fluoride, styrene/acrylonitrile copolymer, polytetrafluoroethylene, polyamide and polystyrene or a mixture thereof; the porous solid support including a thin layer of a reactive polymer deposited thereon for receiving the component to be immobilized; said component being covalently attached to the layer of reactive polymer.

5. The porous, solid support of Claim 4 wherein the component is covalently attached to the reactive polymer on the porous, solid support.

6. The porous, solid support of Claim 5 wherein the reactive polymer is selected from the group comprising chloromethyl¬ styrene/styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.

7. A method of preparing a porous solid support system for the immobilization of a component or components of an immunoassay, said method comprising: providing a porous, solid support having an open pore structure through which liquid can flow; depositing a thin layer of a reactive polymer on the surface area of the porous, solid support; and reacting the component to be immobilized with the layer of reactive polymer to thereby attach said component to the porous, solid support.

8. The method of Claim 7 wherein the immobilized component is covalently attached to the reactive polymer.

9. The method of Claim 8 wherein the reactive polymer is selected from the group comprising chloromethylstyrene/styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.

10. The method of Claim 9 wherein the support is formed from a polymer selected from the group comprising polyethylene, polypropylene, ethylene/vinyl acetate copolymer, polyvinylidene fluoride, styrene/acrylonitrile copolymer, polytetrafluoroethylene, poly and polystyrene.

11. An immunoassay for the analysis of a soluble component in a sample, said assay including passing of said sample through the porous, solid support of Claim 1 whereby said soluble component is bound to the immobilized component on the porous, solid support.

12. The immunoassay of Claim 11 wherein the porous, solid support has a pore size of from about 2 to about 500 microns, the porous solid support including a thin layer of a reactive polymer deposited thereon for receiving the component to be immobilized.

13. The immunoassay of Claim 12 wherein the immobilized component is covalently attached to the reactive polymer on the porous, solid support.

14. The immunoassay of Claim 13 wherein the reactive polymer is selected from the group comprising chloromethylstyrene/ styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.

15. The immunoassay of Claim 14 wherein the porous, solid support for the immobilized component of the immunoassay includes an open pore structure through which liquid can flow, said pores having a pore size of from about 2 to about 500 microns, the solid support being formed from a polymer selected from the group comprising polyethylene, polypropylene, ethylene/vinyl acetate copolymer, polyvinylidene fluoride, styrene/acrylonitrile copolymer, polytetrafluoroethylene, polyamide and polystyrene or a mixture thereof.

16. An immunoassay for the analysis and quantitation of a soluble component in a sample, said immunoassay comprising: providing a porous, solid support having a thin layer of a reactive polymer deposited thereon and a first component of said assay immobilized on said layer of reactive polymer; mixing the sample to be assayed containing a soluble component of the assay to be measured with a second soluble component of the assay; passing the mixture through the porous, solid support whereby the soluble components bind to the immobilized component; and separating the remaining soluble components from the porous, solid support.

17. The immunoassay of Claim 16 wherein the porous, solid support has a pore size of from about 2 to about 500 microns.

18. The immunoassay of Claim 17 wherein the immobilized component is covalently attached to the reactive polymer on the porous, solid support.

19. The immunoassay of Claim 18 wherein the reactive polymer is selected from the group comprising chloromethylstyrene/ styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.

20. An immunoassay for the analysis and quantisation of a soluble component in a sample, said immunoassay comprising: providing a porous, solid support having a thin layer of a reactive polymer deposited thereon; attaching a capture antibody for said soluble component to said reactive polymer whereby it is immobilized thereon; mixing the sample to be assayed containing a soluble component to be measured with a labeled antibody to form a mixture comprising a complex of said soluble component and labeled antibody and unreacted labeled antibody; passing the mixture through the porous, solid support whereby the soluble component binds to the immobilized capture antibody; and measuring the labeled antibody associated with the soluble component bound to the porous, solid support or the unreacted labeled antibody passing through the porous solid support to thereby determine the presence of the soluble component in the sample.

21. The immunoassay of Claim 20 wherein the porous, solid support has a pore size of from about 2 to about 500 microns.

22. The immunoassay of Claim 21 wherein the immobilized component is covalently attached to the reactive polymer on the porous, solid support.

23. The immunoassay of Claim 22 wherein the reactive polymer is selected from the group comprising chloromethylstyrene/ styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.

24. A method of conducting a competitive immunoassay for determining the presence of a soluble component in a sample, said method comprising: providing a porous, solid support having a thin layer of a reactive polymer deposited thereon; affixing a ligand or binder for the soluble substance onto said reactive polymer whereby it is immobilized thereon; forming a reaction mixture comprising a sample suspected of containing the soluble component and a labeled antibody for the soluble component; passing the reaction mixture through the porous, solid support whereby said any soluble component in the sample and labeled antibody bound thereto are bound to the immobilized ligand or binder; and, measuring either the bound or unbound labeled antibody to determine the presence of the soluble component.

25. The immunoassay of Claim 24 wherein the porous, solid support has a pore size of from about 2 to about 500 microns.

26. The immunoassay of Claim 25 wherein the immobilized component is covalently attached to the reactive polymer on the porous, solid support.

27. The immunoassay of Claim 26 wherein the reactive polymer is selected from the group comprising chloromethylstyrene/ styrene copolymer, polyglycidyl methacrylate, polybutadiene oxide, and chlorosulfonated polyethylene.