WO1995034814A1

WO1995034814A1 - Plasma treatment of polymeric materials to enhance immobilization of analytes thereto

Info

Publication number: WO1995034814A1
Application number: PCT/US1995/007500
Authority: WO
Inventors: William N. Black
Original assignee: Abbott Laboratories
Priority date: 1994-06-13
Filing date: 1995-06-13
Publication date: 1995-12-21
Also published as: AU2704995A

Abstract

A method is disclosed for treating the surface of a polymeric material of an assay device to increase the sensitivity of diagnostic assays and screening assays. The method involves the treatment of the surface of the polymeric material with unseparated oxygen plasma to increase the binding capability of an analyte or analyte-binding member to such surface. The treated polymeric material is utilized as a diagnostic assay device or screening assay device for determining the amount or presence of an analyte or analyte-binding member in a test sample.

Description

PLASMA TREATMENT OF POLYMERIC MATERIALS TO ENHANCE IMMOBILIZATION OF ANALYTES THERETO

Field Of The Invention

The present invention relates to a method of treating the surface of an assay device with an unseparated plasma for increasing the sensitivity of the assay device. In particular, the present invention relates to the use of ionized oxygen to treat the surface of an assay device comprising a polymeric material in order to enhance the immobilization of an analyte or binding member thereof to the surface of the assay device.

Background Of The Invention

In the diagnosis and treatment of disease, it is of interest to determine whether a particular substance is present in biological fluids or other materials by various diagnostic methods known in the art. It is also of interest to determine whether a particular substance is present in transplant tissues, such as whole blood, plasma, organs, and the like, by various screening methods known in the art. Such substances are commonly referred to as "analytes" and include, but are not intended to be limited to, antibodies, antigens, haptens, and the like. Various analytical procedures and devices are commonly employed in diagnostic and screening assays to monitor and quantitate such analytes. Such analytical procedures typically utilize immobilized reagents such as antibodies, antigens, enzymes, carbohydrates, nucleoti.de sequences, and the like. An important consideration in these assays is the quantity and uniformity with which these reagents can be immobilized or bound to a particular surface area. Increased reagent immobilization or binding can provide greater sensitivity of the assay and decrease the required volume of the test sample. The importance of reagent binding to a solid surface has stimulated the development of a variety of methods to increase the binding of proteins to solid surfaces. Each method offers advantages and disadvantages for the binding of certain analytes. For example, U.S. Patent No. 4,973,493 discloses a method for the direct chemical modification of protein binding surfaces. U.S. Patent Nos. 5,034,428 and 4,829,098 disclose pre-irradiation of a polymeric surface with ionizing radiation at -78 ^#C to increase protein binding to the irradiated surface. U.S. Patent No. 4,919,659 discloses a method of coating a polymer onto a supporting surface and then treating that surface with an organic plasma gas to create an activated surface for increased fibronectin binding. European Patent Application Publication No. 0 076 562 teaches a method for grafting specific chemical functional groups onto a substrate surface by contacting such surfaces with selected components of a plasma of a vaporized material.

In addition, plasma treatment methods have been used in industry to change surface properties of different materials for various commercial applications. For example, a variety of gases and plasma operating parameters have been used to treat diverse materials in order to clean the surface of organic contamination, remove material by ablation, crosslink surface molecules, and modify surface chemistry. Plasmas are defined as highly reactive and energetic mixtures of ions, electrons and photons with properties distinct from ordinary gases. Four types of man-made plasmas which are available are thermal plasmas, discharge plasmas, beam plasmas and hybrid plasmas, such as corona and ozonizer-type discharges. Electrical discharge plasmas are usually generated by the use of either DC sources or AC sources of a frequency through the microwave range at power levels from about 1.0 W to about 10 kW.

Plasma free radicals have sufficient chemical energy to break bonds in surface molecules. For example, the lowest excited state for the oxygen radical is 220 Kcal/mole above the ground state, which is sufficient to break almost any organic bond. Thus when plasma free radicals impinge on a polymeric surface, they have sufficient energy to break chemical bonds in that surface and abstract atoms or molecular fragments. This abstraction process leaves residual free radicals in that surface. These surface radicals can either react with themselves or react with the plasma gas to form new chemical species in that surface. Although efforts to increase the binding of analytes to solid surfaces have been made, the available methods are either unsuitable for biomolecules, such as analytes (e.g., denaturation of protein), or are commercially prohibitive in that such methods require multiple steps, multiple reagents, or expensive sophisticated equipment. For example, the method disclosed in U.S. Patent No. 4,919,659 requires that a polymer be precoated on a supporting surface whereby surface uniformity and characteristics are dependent upon a polymer coating step and a plasma treatment step.

The method disclosed in European Patent Application Publication No. 76,562 requires the separation of plasma components before treating the substrate surface. However, not only does this separation of plasma components require additional equipment to perform, but it provides for the grafting of specific chemical functional groups on the polymer surface for binding biomolecules. When immobilizing analytes such as antibodies and antigens, it is often preferable to provide diverse chemical functional groups for interaction so that the immobilized molecule will be available in multiple conformations. This is particularly important if one is immobilizing, for example, an antiserum fraction as the active analyte-binding member.

Accordingly, a need exists for an economical, commercially viable means of increasing surface binding potential for analytes and other biomolecules, such as analyte-binding members, on polymeric surfaces that can be used to increase the sensitivity of polymeric assay devices.

Summary Of The Invention

The present invention provides methods for attaching increased quantities of an analyte or analyte-binding member to polymeric surfaces. The invention advantageously employs an unseparated plasma treatment of the surface of a polymeric material to enhance the immobilization of biomolecules thereto, such as the immobilization of an analyte or analyte-binding member to the surface of such polymeric material.

According to one embodiment of the present invention, a method for increasing the sensitivity of a diagnostic assay or screening assay is provided whereby the surface of a polymeric material of an assay device is activated with an unseparated oxygen plasma, and an analyte or analyte-binding member is then immobilized to such activated polymeric surface.

According to another embodiment of the present invention, a diagnostic assay device or screening assay device comprising a polymeric material that has been pre-treated with unseparated oxygen plasma, and having an analyte or analyte-binding member immobilized or bound to the polymeric material, is provided.

According to still another embodiment of the present invention, a method for immobilizing an analyte or analyte-binding member to the surface of a polymeric material for use as a diagnostic assay device or screening assay device is provided comprising the steps of: 1) placing the polymeric material in a vacuum environment;

2) introducing substantially pure oxygen into the vacuum environment;

3) ionizing the oxygen within the vacuum environment;

4) allowing the polymeric material to react with an unseparated oxygen plasma to give a treated polymeric surface;

5) removing the reacted polymeric material from the vacuum environment; and

6) contacting an analyte or analyte-binding member with the treated polymeric surface. According to yet another embodiment of the present invention, a diagnostic or screening assay device comprising a polymeric surface that has been activated with an unseparated oxygen plasma and an analyte or analyte-binding member immobilized on the activated polymeric surface is provided.

Accordingly, the present invention provides an economical, commercially viable means of increasing surface binding capability for an analyte or analyte-binding member to the surface of a polymeric material. Furthermore, the present invention provides a means of increasing the uniformity of distribution of an analyte or analyte-binding member to the surface of a polymeric material. Still further, the present invention increases the sensitivity of screening assays and diagnostic assays, and, in some instances, may decrease the required sample size for such assays by increasing analyte or analyte-binding member binding to the surface of a polymeric material of a screening assay device or a diagnostic assay device. Moreover, the present invention provides a reproducible method, suitable for the commercial manufacturing of screening assay devices or diagnostic assay devices, for increasing the immobilization of analyte or analyte-binding member to the surface of a polymeric material, enable the development of screening assays and diagnostic assays for the detection of analytes or analyte-binding member present in very low concentrations in biological fluids, and to increase the accuracy of screening assays and diagnostic assays.

Brief Description Of The Drawings

Figure 1 is a block diagram of a plasma reactor.

Figure 2 is a perspective view of a vacuum chamber.

Figure 3 is a top view of a treated polymeric multiwell immunoassay tray.

Figure 4 is a graphical representation of a monoclonal antibody bound to oxygen gas plasma treated multiwell plates and untreated multiwell plates as measured in hepatitis surface antigen subtype-ad sensitivity and goat anti-mouse antibody absorbances.

Figure 5 is a graphical representation of monoclonal antibody bound to oxygen gas plasma treated and untreated multiwell plates as measured in hepatitis surface antigen subtype-ay sensitivity and goat anti-mouse antibody absorbances.

Definitions

The term "test sample", as used herein, refers to a material suspected of containing analyte or analyte-binding member. The test sample can be used directly as obtained from the source or following a pretreatment to modify the character of the sample. The test sample can be derived from any biological source, such as a physiological fluid, including, blood, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, raucous, synovial fluid, peritoneal fluid, amniotic fluid, and the like. The test sample can be pretreated prior to use, such as preparing plasma from blood, diluting viscous fluids, and the like. Methods of treatment can involve filtration, distillation, concentration, inactivation of interfering components, and the addition of reagents. Besides physiological fluids, other liquid samples can be used such as water, food products, and the like, for the performance of environmental or food production assays. In addition, a solid material suspected of containing the analyte or analyte-binding member can be used as the test sample, such as organ tissue, and the like. In some instances, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte or analyte- binding member. The term "analyte or analyte-binding member," as used herein, refers to the compound or composition to be detected or measured and which has at least one epitope or binding site. The analyte or analyte- binding member can be any substance for which there exists a naturally occurring binding member or for which a binding member can be prepared. Analytes or analyte-binding members include, but are not intended to be limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), virus particles, and metabolites of or antibodies to any of the above substances. In particular, such analytes or analyte-binding members include, but are not intended to be limited to, ferritin; creatinine kinase MIB (CK-MB); digoxin; phenytoin; phenobarbital, carbamazepine; vancomycin; gentamicin; theophylline; valproic acid; quinidine; leutinizing hormone (LH); follicle stimulating hormone (FSH); estradiol, progesterone; IgE antibodies; vitamin B2 micro-globulin; glycated hemoglobin (Gly. Hb); cortisol; digitoxin; N-acetylprocainamde (NAP A); procainamide; antibodies to rubella, such as rubella-IgG and rubella-IgM; antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylates; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies to hepatitis B core antigen, such as anti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immune deficiency virus 1 and 2 (HIV-1 and HIV-2); human T-cell leukemia virus 1 and 2 (HTLV-1 and HTLV-2); hepatitis B envelope antigen (HBeAg); antibodies to hepatitis B envelope antigen (Anti-HBe); thyroid stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (Total T3); free triiodothyronine (Free T3); carcmoembryoic antigen (CEA); and alpha fetal protein (AFP). Drugs of abuse and controlled substances include, but are not intended to be limited to, amphetamine; methamphetamine; barbiturates such as amobarbital, secobarbital, pentobarbital, phenobarbital, and barbital; benzodiazepines such as librium and valium; cannabinoids such as hashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone and opium; phencyclidine; and propoxyhene. The term "analyte or analyte-binding member" also includes any antigenic substances, haptens, antibodies, macromolecules and combinations thereof.

The term "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 or analyte-binding member itself. The analyte-analog can include a modified analyte or analyte-binding member as well as a fragmented or synthetic portion of the analyte or analyte-binding member molecule, so long as the analyte-analog has at least one epitopic site in common with the analyte or analyte-binding member of interest. An example of an analyte-analog is a synthetic peptide sequence which duplicates at least one epitope of the whole-molecule analyte or analyte-binding member so that the analyte-analog can bind to an analyte-specific binding member.

The term "binding member", as used herein, refers to a member of a binding pair, i.e. two different molecules wherein one of the molecules specifically binds to the second molecule through chemical or physical means. In addition to antigen and antibody binding pair members, other binding pairs include, but are not intended to be limited to, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g. ribonuclease, S-peptide and ribonudease S-protein), and the like. Furthermore, binding pairs can include members that are analogs of the original binding member, for example, an analyte-analog or a binding member made by recombinant techniques or molecular engineering. If the binding member is an immunoreactant it can be, for example, a monoclonal or polyclonal antibody, a recombinant protein or recombinant antibody, a chimeric antibody, a mixture(s) or fragment(s) of the foregoing, as well as a preparation of such antibodies, peptides and nucleotides for which suitability for use as binding members is well known to those skilled in the art. Detailed Description of the Invention

The present invention provides a method of treating the surface of a polymeric material with an unseparated plasma for use thereof as a diagnostic assay device or screening assay device. The method of the present invention increases the sensitivity of screening assays or diagnostic assays by increasing the concentration of analyte or analyte-binding member immobilized to the treated surface of such assay device. In particular, the present invention relates to a method of treating the surface of a polymeric material with an unseparated oxygen plasma to increase the quantity of analyte or analyte-binding member immobilized thereon. The phrase "surface of a polymeric material" and the phrase "polymeric surface" are used synonymously. The term "unseparated" is intended to mean that the entirety of the plasma components are used as a whole without any separation or any selection.

Suitable assay devices, such as, for example, multiwell plates, beads, microparticles, test tubes, microscope slides, dipsticks, and the like, may be manufactured from different types of polymeric organic material including, but not intended to be limited to, polyethylene acetate, polypropylene acetate, polystyrene, and the like. However, it is to be understood that other forms of plastic, elastomers, fibers, and the like, can be treated according to the present invention to provide an activated surface for increased binding of analytes or analyte-binding member and other assay reagents. Further, it is possible to coat the surface of an assay device made from an inorganic material with a layer of organic polymeric material. The inorganic material can be, for example, glass, metal, and the like.

The treatment of the surface of a polymeric material or polymeric surface with oxygen plasma, such as ionized oxygen, activates the surface of the polymeric material to thereby increase both the hydrophilicity and the surface tension of the polymeric surface. These changes in the surface characteristics of the polymeric material significantly improve the uniformity, the quantity and the strength of binding of an analyte or analyte-binding member to the surface of the polymeric material. For example, the present inventor has unexpectedly and surprisingly found that these changes in the binding characteristics of the activated polymeric surface or substrate are stable for at least about one (1) year.

The assay device according to the present invention can be utilized in various heterogeneous immunoassay formats known in the art for the determination of an analyte or analyte-binding member in a test sample in various diagnostic and screening assays. Generally, such heterogeneous immunoassay formats involve a labeled reagent or tracer comprising an analyte or analyte-binding member, an analog of the analyte or analyte-binding member, or an antibody thereto, labeled with a detectable moiety or component, to form a free spedes and a bound species. In order to correlate the amount of tracer in one of such spedes to the amount of analyte or analyte-binding member present in the test sample, the free spedes must first be separated from the bound spedes, which can be accomplished according to methods known in the art employing solid phase materials, such as an assay device according to the present invention. Such solid phase materials are utilized for the direct immobilization of one of the binding participants in the binding reaction, such as the antibody, analyte or analyte-binding member or analog of the analyte or analyte-binding member. For example, one of the binding partidpants can be immobilized on the surface of a solid phase material such as a test tube, beads, partides, microparticles or the matrix of a fibrous material, and the like, according to methods known in the art.

Heterogeneous immunoassays can be performed in a competitive immunoassay format wherein, for example, the antibody can be immobilized to a solid phase material whereby upon separation, the amount of the tracer which is bound to such solid phase material can be detected and correlated to the amount of analyte or analyte- binding member present in the test sample. Another form of a heterogeneous immunoassay employing a solid phase material is referred to as a sandwich immunoassay, which involves contacting a test sample containing, for example, an antigen with a protein, such as an antibody or another substance capable of binding the antigen, and which is immobilized on a solid phase material. The solid phase material typically is treated with a second antigen or antibody which has been labeled with a detectable moiety. The second antigen or antibody then becomes bound to the corresponding antigen or antibody on the solid phase material and, following one or more washing steps to remove any unbound material, an indicator material, such as, for example, a chromogenic substance which reacts with the detectable moiety (e.g., where the detectable moiety is an enzyme, a substrate for such enzyme is added) to produce a color change. The color change is then detected and correlated to the amount of antigen or antibody present in the test sample.

According to one embodiment of the present invention, a method to enhance the immobilization of an analyte or analyte- binding member to the surface of a polymeric material for use thereof as a diagnostic assay or screening assay device is provided, wherein the method comprises the steps of:

1) placing the polymeric material in a vacuum environment of from between about 0.2 Torr and about 1.0 Torr;

2) introdudng substantially pure oxygen into the vacuum environment;

3) ionizing the oxygen within the vacuum environment with a high frequency radiation of from between about 20 Hz and about 60 Hz;

4) allowing the polymeric surface to react with an unseparated oxygen plasma to give a treated surface of polymeric material in a time period of from between about 0.5 minute and about 10 minutes;

5) removing the reacted polymeric material from the vacuum environment; 6) contacting an analyte or analyte-binding member with the treated polymeric surface at a temperature of from between about 2° and about 40°C for a time period of from between about 1 minute to about 75 minutes;

7) removing unbound analyte or analyte-binding member from the surface of the polymeric material;

8) washing, with a buffer solution at a temperature of from between about 2°C and about 40°C for a time period of from between about 30 seconds to about 2 hours the polymeric material whose surface having attached thereto the analyte or analyte-binding member;

9) dispensing an overcoat solution to the washed polymeric material; and 10) drying the resultant polymeric material.

The invention is further defined by reference to the following examples, which are intended to be illustrative and not limiting.

Example 1 Activation of Multiwell Plate Surface with Ionized Oxygen Multiwell plates were treated with an oxygen plasma such that the hydrophilidty and surface tension was increased thereby. The plates were treated utilizing a plasma reactor (FIGURE 1) consisting of vacuum pump 10, gas manifold 11, oxygen source 12, power supply 13, and vacuum chamber 20. Vacuum pump 10 was preferably capable of evacuating the system to less than about 0.5 Torr in about one (1) minute or less. Gas manifold 11 controlled the pressure/flow of gas and/or vacuum into and out of vacuum chamber 20. Oxygen source 12 could be a cylinder of commerdally available pure oxygen gas. Power supply 13 furnished the power required to generate the plasma. Energy in the form of high-frequency radiation (about 40 KHz) was supplied by an RF generator to the electrodes in order to create plasma in the processing chamber. Preferred power supply 13 generates radio frequency (RF) plasmas at about 13.56 MHz.

The preferred plasma reactor was designed for batch operation. FIGURE 2 shows a perspective view of vacuum chamber 20. Vacuum chamber 20 was designed to be loaded with a batch of multiwell plates 30 (as illustrated in FIGURE 3).

Multiwell plate 30 was placed on vacuum chamber tray shelf 21. Once vacuum chamber 20 had been loaded with multiwell plates 30, vacuum chamber door 22 was secured and the vacuum chamber was evacuated to about 0.3 Torr.

Oxygen was then introduced into vacuum chamber 20 through gas port 23. Power supply 13, a RF energy generator, was activated. The oxygen gas was ionized when it encountered energized vacuum chamber electrodes 24. The oxygen gas was generally ionized within a few minutes.

Ionized oxygen was then allowed to diffuse through the chamber and to react with multiwell plates 30. Once the reaction between ionized oxygen and the reaction surface of multiwell plates 30 was completed, the vacuum chamber was purged with nitrogen and the pressure was equalized with atmospheric pressure.

Vacuum chamber door 22 was opened and the adivated multiwell plates were unloaded.

After about one (1) minute, the treatment was usually completed. To assure that all trays were treated, the trays were generally treated for about 5-10 minutes. After the treatment, the hydrophobic polymeric material or substrate became very hydrophilic. Example 2 Immobilization of Analyte-Binding Member to Multiwell Plate Analyte-binding member was bound to an activated surface of multiwell plate (Curtesy Plastics, Inc.). Multiwell plates 30 were commonly used in diagnostic assay kits.

Each individual well 31 of activated multiwell plate 30 was filled with approximately 0.2 ml of an antiserum, for example anti-HBS. An equal volume of a 15mM propylamine solution was then added to each well and multiwell plate 30 was leveled to ensure uniform dispersion of the solutions. Multiwell plate 30 was incubated at room temperature with the antiserum solution for 1-2 hours before the antiserum solution was aspirated.

Individual well 31 of multiwell plate 30 was then filled with a buffered wash solution and leveled. The wash solution was incubated at about 40 *C for 90 minutes before aspiration.

Finally, an overcoat solution was dispensed into the wells and multiwell plate 30 leveled. Then, the overcoat solution was incubated in multiwell plate 30 at 40 ^»C for 30 minutes. Then, the overcoat solution was aspirated and multiwell plate 30 was dried and packaged.

Example 3

Comparison of Monoclonal Antibodies Bound to Plasma Treated and Untreated Multiwell Plates Method: Two experiments were completed to demonstrate the efficiency of binding monoclonal antibodies (mouse IgM anti-HBsAg) to gas plasma treated and untreated multiwell plates 30. In the first 1 8

experiment, antibodies bound to the multiwell plates captured Hepatitis B Surface Antigen (HBsAg) from HBsAg positive plasma panel members. Captured antigen was tagged with goat polyclonal antibodies to HBsAg conjugated with horseradish peroxidase (anti-HBs:HRPO). In the second experiment, polyclonal anti-mouse antibodies conjugated with horseradish peroxidase (IgM specific, anti-mouse:HRPO) labeled the multiwell plate immobilized mouse monoclonal antibodies. In both experiments, the HRPO-conjugated antibody present on the multiwell plate was quantitated by adding O- Phenylenediamine, a substrate for HRPO, and measuring color development using the Abbott Parallel Processing Center (PPC; Abbott Laboratories, Abbott Park, IL, USA). PPC is a tray processing system that washes added reagents and read 460nm color development for blood screening purposes. The multiwell plates were oxygen gas plasma treated before reacting the multiwell plates with mouse IgM anti-HBsAg. Multiwell plates were gas plasma treated by loading them into a gas plasma reador and sealing the door. Then, the reactor was evacuated to about 0.3 Torr and filled with pure oxygen. Energized vacuum chamber electrodes ionized the oxygen gas which diffused through the diamber and reacted with the multiwell plates for 10 minutes. The vacuum chamber was purged with nitrogen and re-equalized to atmospheric pressure before opening the chamber door.

Wells of the gas plasma treated and untreated multiwell plates were coated with monoclonal mouse IgM anti-HBsAg diluted in 15mM MES and 0.2% Tween 20 buffer (pH=5.5). The diluted antibody solution (200μL) was further diluted in the well with 200μL of 0.1M propylamine in 15mM MES and 0.2% Tween 20 (pH=5.5). This mixture was mixed by rotating the multiwell plate at 200 revolutions per minute (rpm) at room temperature for 30 minutes before aspirating. Then, 400μL of a phosphate buffered saline containing 0.5% Tween 20 (pH=7.3) was added to each well. After incubating for 90 minutes at 40 *C, the wash solution was aspirated and 400μL of an overcoat solution (0.1M sodium citrate buffer, containing 3% bovine serum albumin and 5% sucrose, pH=7.3) was added to each well. The overcoat solution was aspirated after 30 minutes and the multiwell plates were dried at room temperature overnight.

The quantity of monoclonal antibody bound to oxygen gas plasma treated and untreated multiwell plates were measured by the difference in HBsAg sensitivities and goat anti-mouse absorbances. Sensitivity being the lowest detectable level of HBsAg in ng/ml. The sensitivities were calculated by linear regression of a diluted positive

HBsAg ad and ay plasma panel. Concentrations of the plasma panel members (Abbott Catalog Number 9108, Abbott Laboratories, Abbott Park, IL, U.S.A.) were determined by Auszyme Monoclonal using linear regression. The absorbance values (A460) for each panel member are plotted on the X axis and verses it's concentration (ng/mL) on the Y axis. The slope of the line is calculated and the sensitivity determined (lowest detedable level of antigen). The sensitivity using linear regression uses the equation of a line.

y = cutoff at the Y-axis m = slope x = sensitivity (ng/mL) b = Assay background, negative control To determine the difference in HBsAg sensitivities, the Abbott Multiscreen® Test Procedure (Abbott Laboratories, Abbott Park, IL, U.S.A) was used to test the treated and untreated multiwell plates. Negative plasma (human plasma that is non-reactive for Hepatitis B surface antigen and antibodies to HIV-1 and HIV-2), HIV-1 positive plasma (human plasma positive for HIV-1 antibodies) and HLV-2 positive plasma (human plasma positive for HIV-2 antibodies) controls were induded in each experiment to validate the experimental procedure. The plasma panel used to determine sensitivities is described above (Abbott catalog number 9108). A 150μL sample of negative, FLIV-l positive, HIV-2 positive controls and the HBsAg positive plasma panel members was added to the appropriate gas plasma treated and untreated wells of the multiwell plates. Goat anti-HBs:HRPO (50μL) was added to each well and mixed with the plasma samples. To this mixture, one recombinant HIV-1 p24, HIV-1 p41 and HTV-2 p36 coated bead was added to each well. Trays were incubated to allow antibodies to HTV-1 and fflV-2 to bind to the HTV-l p24, HIV-1 p41 and fflV-2 p41 antigen coated beads. Also, the incubation allowed Hepatitis B surface antigen to bind to the mouse monodonal anti-HB3Az bound to the tray. The trays were rotated during the assay incubations at an elevated temperature of 40°C (Abbott Dynamic Incubator, DI, Abbott Laboratories, Abbott Park, IL, U.S.A.) for 120 minutes. Then, the PPC was used to wash the mixture from the wells and added 200μL of Multiscreen Wash Reagent (Abbott Laboratories, Abbott Park, IL, U.S.A), to wash or remove potentially interfering substances. After the trays were rotated (DI) for 30 minutes at 40 »C, the wells were washed by the PPC and 200μL of HTV-1 p24, fflV-2 p41 and HTV-2 p36 conjugate to label bound HIV-1 and HIV-2 antibodies, reaction would color if conjugate antibodies bind to bound antibodies was added to each well. After rotating for 30 minutes at 40 »C, the HIV conjugate was washed from the wells and 300μL of o- Phenylenediamine (OPD) substrate was added. The OPD was incubated in the well for 30 minutes at room temperature and was quenched with 300μL of IN sulfuric acid. Bound mouse monoclonal antibodies were also detected directly with anti-mouse:HRPO conjugate. A 200μL sample of conjugate (diluted 1:10,000 in phosphate buffered saline (PBS)) was added to five wells of treated and five wells of untreated multiwell plates. After rotating (DI) at 40 «C for 30 minutes, the conjugate was washed from the wells and 300μL of OPD substrate was added. The OPD was incubated in the wells for 30 minutes at room temperature before the reaction was quenched with IN sulfuric add.

For both experiments, the absorbances were measured at 460nm using the PPC. Results: The determined absorbances are listed in Table 1. The

HBsAg subtypes ad and ay calculated sensitivities for both treated and untreated multiwell plates are listed in Table 2. Table 1

Treated Untreated Experimental Controls *Net A460 *Net A460 Negative Human Plasma (not net O.D.) 0.027 0.027

Diluted Plasma Positive for fflV-l antibodies 0.760 0.757

Diluted Plasma Positive for HBsAg 0.967 0.154

Diluted Plasma Positive for HIV-2 antibodies 0.409 0.428

Abbott Multiscreen Cutoff Calculation (0.10 + **Negative Control) Cutoff (not net O.D. cutoff) 0.127 0.127

Net Cutoff (Cutoff - Negative Control) 0.100 0.100

Diluted Positive HBsAg subtype ad Plasma Panel Members 9108-015 ad B (1.55ng/mL) 0.317 0.008

9108-015 ad D (0.74ng/mL) 0.146 0.005 9108-015 ad E (0.55ng/mL) 0.119 0.023

9108-015 ad G (0.27ng/mL) 0.071 0.021

Diluted Positive HBsAg subtype ad Plasma Panel Members 9108-015 ay B (1.65ng/mL) 0.484 0.011

9108-015 ay D (0.84ng/mL) 0.239 0.004 9108-015 ay E (0.64ng/mL) 0.168 0.007

9108-015 ay G (0.35ng/mL) 0.099 0.012

* Net is the A460 absorbance value subtracting the negative human plasma value (Negative Control). ** Negative Control is the same as experimental control negative human plasma.

Table 2 Calculated HBsAg subtypes ad and ay Sensitivities

Treated Untreated subtype ad Sensitivity (ng/mL) 0.454 *** subtype ay Sensitivity (ng/mL) 0.379 ***

*** Because the calculated slopes are either negative or zero, the sensitivities cannot be calculated.

The absorbances for HBsAg subtypes ad and ay plasma panels were plotted (FIGURE 4 and 5) against the concentration of HBsAg (ng/ml). Using a linear regression analysis, the sensitivities were determined (Table 2). For the treated multiwell plate, the slope of the HBsAg ad and ay lines were 0.194 and 0.301 with correlations of 0.997 and 0.999, respectively. The untreated multiwell plate sensitivities could not be determined because the slope of the HBsAg ad line is negative and the HBsAg ay line is zero.

The absorbances for the treated and untreated multiwell plates after reacting with goat anti-mouse:HRPO are listed in Table 3. The signal (A460) from the untreated tray is fifteen times lower than that of the oxygen gas plasma treated tray.

Table 3

Goat Anti-Mouse:HRPO Absorbances # of Wells Tested Mean A460 Untreated Multiwell Plate 5 0.112 Oxygen Gas Plasma Multiwell plates 5 1.680

Oxygen gas plasma treatment of the multiwell plates greatly improved the binding of mouse monodonal anti-HBs antibodies to the polystyrene substrate of the plate surfaces. Antibody molecules were permanently bound or immobilized to plasma-oxygen-treated surface of polymeric materials; whereas without plasma oxygen adivation, antibody molecules were only weakly bound, if at all.

Although the present invention and its advantages have been described in detail, it should be appredated by those skilled in the art that the conception and the specific embodiment disdosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating the surface of a polymeric material, said method comprising the steps of: (a) activating said polymeric surface of said assay device with an unseparated oxygen plasma to provide an activated polymeric surface; and

(b) immobilizing an analyte or analyte-binding member to said activated polymeric surface.

2. The method of daim 1, wherein said polymeric surface is selected from the group consisting of the surface of a well of a multiwell plate, the surface of a dip stick assay device, the surface of a bead, and the surface of a micropartide.

3. The method of daim 1, wherein said analyte or analyte- binding member is seleded from the group consisting of nudeotides, carbohydrates, and haptens.

4. The method of daim 1, wherein said analyte or analyte- binding member comprises a protein.

5. The method of daim 1, wherein said analyte or analyte- binding member comprises an enzyme.

6. The method of daim 1, wherein said analyte or analyte- binding member comprises an antibody.

7. The method of daim 1, wherein said analyte or analyte- binding member comprises an antigen.

8. An assay device prepared according to the method of claim 1.

9. A method for immobilizing an analyte or analyte-binding member to the surface of a polymeric material, said method comprising the steps of: placing said polymeric material in a vacuum environment; introducing essentially pure oxygen into said vacuum environment; ionizing said oxygen within said vacuum environment; allowing said polymeric material to read with said ionized oxygen to provide a reacted polymeric surface; removing said reacted polymeric material from said vacuum environment; and immobilizing an analyte or analyte-binding member to said readed polymeric surface.

10. The method of daim 9, wherein said surface of said polymeric material is selected from the group consisting of the surface of a well of a multiwell plate, the surface of a bead, the surface of a micropartide, and the surface of a dip stick assay device.

11. The method of daim 9, wherein said analyte or analyte- binding member is seleded from the group consisting of nudeotides, carbohydrates, and haptens.

12. The method of daim 9, wherein said analyte or analyte- binding member comprises a protein.

13. The method of claim 9, wherein said analyte or analyte- binding member comprises an enzyme.

14. The method of daim 9, wherein said analyte or analyte- binding member comprises an antibody.

15. The method of daim 9, wherein said analyte or analyte- binding member comprises an antigen.

16. An analyte or analyte-binding member immobilized to a surface of polymeric material according to the method of claim 9.

17. An assay device comprising: a polymeric material having a surface treated with an unseparated oxygen plasma; and an analyte or analyte-binding member immobilized on said treated surface of said polymeric material.

18. The assay device of claim 17, wherein said polymeric material is selected from the group consisting of polyethylene acetate, polypropylene acetate, and polystyrene.

19. The assay device of claim 17, wherein said assay device is selected from the group consisting of multiwell plates, dipsticks, slides, beads, and micropartides.

20. The assay device of claim 17, wherein said analyte or analyte-binding member is selected from the group consisting of nudeotides, carbohydrates, and haptens.

21. The assay device of claim 17, wherein said analyte or analyte-binding member comprises a protein.

22. The assay device of claim 17, wherein said analyte or analyte-binding member comprises an enzyme.

23. The assay device of claim 17, wherein said analyte or analyte-binding member comprises an antibody.

24. The assay device of claim 17, wherein said analyte or analyte-binding member comprises an antigen.