GB2194046A - Solid phase binding reagents, preparation thereof and assay kits containing same - Google Patents

Description

SPECIFICATION

BACKGROUND OF THE INVENTION Solid phase binding reagents, preparation thereof and assay kits containing same This invention relates to biochemical binding assays of the heterogeneous type, i.e., those involving biochemical binding between species immobilized on a solid phase support and species dissolved or suspended in liquid. In particular, this invention relates to solid phase reagents comprising supports having affixed thereto in a distinct pattern a plurality of binding species derived from a common source such as a cell or viral lysate. The analysis of biological fluids for certain species present in combination is often done for diagnostic or screening purposes. Many such assays involve incubating a sample of the fluid with a strip of solid phase material to which is bound a series of species each having a binding affinity for one of the species sought to be detected. The strip is then read to detect the presence of binding between species originally on the strip and those in the sample. The species immobilized on the strips are typically such biochemical species as proteins or peptides which are characteristic components of a larger biochemical species such as a virus or cell. In the past, such speciesbound supports have been blots of electropherograms. Since a single electropherogram can generate only a limited number of such blots, the blots have inherent limitations in terms of accuracy, reproducibility and overall reliability. Variations from one electrophoretic separation to the next, as well as from one starting mixture to the next are inevitable. The use of electropherograms further limits the degree to which the various species may be separated from each other and focused into well defined bands.Variations in the amounts of the various species in the starting mixture will raise further problems by creating an over-emphasis of certain species at the cost of others, thereby making detection of the entire mixture difficult.

SUMMARY OF THE INVENTION It has now been discovered that solid phase binding reagents which provide a high degree of accuracy, readability and reproducibility in a binding assay may be prepared from a biological source mixture by isolating the various binding species of choice from the mixture, diluting each isolated species in a carrier liquid to form a series of stock mixtures each containing one of the isolated species at a preselected concentration, and applying the mixtures to discrete, precisely defined regions on a solid phase support to immobilize the various species on the support according to a preselected pattern. The invention has particular utility in preparing a multitude of such patterns on separate supports in identical manner, where all species form sharply defined bands or binding regions of substantially uniform density.Such supports may be incorporated into assay kits containing materials for performing identical assays on a multitude of samples, such kits further containing additional components required for the performance of the assay.

DETAILED DESCRIPTION OF THE INVENTION

AND PREFERRED EMBODIMENTS Biological source mixtures useful in the practice of the present invention may vary widely in composition and source. Such mixtures will generally include any combination of biological species, particularly macromolecules, which occur as mixtures, clusters or combinations. The invention has particular utility to mixtures of such species as peptides, polypeptides, and proteins. Typical examples of such mixtures are cell and viral lysates. The invention may be used for instance to isolate and separate cell or viral antigens to use as binding species to detect the presence of antibodies to the antigens in a sample of bodily fluid. Alternatively, the species in the mixture may be the antibodies themselves, to be isolated and used for detecting the presence of antigens in the fluid sample. In essence, the species of the mixture to be separated in accordance with the present invention will be any combination of species each of which has a specific binding affinity for one of several components suspected to be present in the test sample. One specific use contemplated by the inventors herein is the preparation of a solid phase binding reagent for an assay for detecting the presence of antibodies to one or more of the viruses associated with Acquired Immune Deficiency Syndrome (AIDS). Examples of recognized AIDS viruses are Human T-Cell Leukemia Virus-))! (HTLV-III), Lymphadenopathy Associated Virus (LAV) and Acquired Immune Deficiency Syndrome Related Virus (ARV). Prior to the separation of the species, the mixture is generally prepared and processed according to conventional techniques. For cells and viruses, particularly viruses residing in cell cultures, the cells and viruses are in some cases killed and lysed as part of the preparation, preferably by a combination of detergent and heat, in such a manner that the binding properties of the antigens or other species to be used in the assay are not substantially altered. The separation and isolation of the binding species from the source mixture is done by a separation process or sequence tailored to the starting mixture and to the species sought to be recovered. In many cases, it will be desirable to isolate certain species to the exclusion of others, the selected species being those which bind to the species in the sample whose presence is most indicative of the condition sought to be detected. The identity of the most indicative species in a sample in terms of a given condition will be known to those skilled in the art. Separation and isolation may be achieved by conventional preparative separation techniques, such as the various forms of chromatography used in biochemical preparations. Depending on the extent and variety of species sought, a sequence of separations of various types may be necessary to isolate each one. One or more of such techniques as high performance liquid chromatography, reversed-phase chromatography, gel permeation chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and size exclusion chromatography may be included in the sequence. Each binding species of interest in the source mixture must be fully separated from each of the other such species, but complete isolation of the species from all other components is not always necessary. In the case of viral proteins originating in a virus grown in a particular cell line, for example, contaminating proteins and nucleic acids from the host cells will also be present. If these are not immunoreactive with the same antibodies which bind to the viral proteins, they need not be completely removed. It is preferred, however, that a sufficient quantity of these and other extraneous materials be removed such that the species of interest comprise at least about 80 to 90% of the isolate. In selecting conditions for the isolation procedures, one must take into account the need for retaining the binding capabilities (e.g., the immunoreactivity) of the individual species. Membrane proteins for instance are frequently very hydrophobic, requiring severe conditions such as high concentrations of detergents or organic solvents for elution from a particular column. These potentially harsh conditions can denature proteins to the point that they no longer react with antibodies to the native protein. The best procedures therefore will be a balance between denaturing conditions with high recovery and milder conditions with greater immunoreactivity. Once isolated from the source mixture, the species are individually diluted in carrier liquids to form individual stock solutions each containing one or approximately one of the species. In some cases, a particular species may be defined as a narrow molecular weight range and may correspond to a relatively broad electropherogram band with blurred boundaries, while other species may be defined as single molecular weights. Dilution may involve either dissolving or suspending the species in the carrier liquid, depending on its solubility. The carrier liquid itself may be aqueous or organic or a mixture, and will preferably be a buffer solution. Examples include phosphate-buffered saline (PBS), TRIS/methanol, PBS/propanol, PBS/acetonitrile and carbonate. The concentration of the species in the carrier liquid is not critical and may vary widely.The concentration will generally be selected, however, with a view toward the species density sought to be achieved in its final condition as bound to the solid phase support. In most applications, a concentration ranging from about 0.01 to about 10 micrograms per milliliter will provide the best results. It will frequently be desirable to include additional components in the stock solutions for a variety of reasons. Examples include detergents such as sodium dodecylsulphate (SDS), Triton X-100 (Rohm & Haas Co., Philadelphia, Pennsylvania), 3-[chloramidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS), and Tween 20, 40, 60 and 80 (Atlas Chemical Industries, Inc., Wilmington, Delaware). Bactericidal and bacteristatic agents may also be included--examples are sodium azide and thimerosal. If the species are to be covalently bound to the support, various coupling agents known among those skilled in the art may be used. Examples include EDAC (ethyl dimethylaminopropyl carbodiimide) and bifunctional reagents such as, for example, MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester). These coupling agents are generally not included in the stock solution, and their method of use may follow conventional procedures. For example, the entire support surface may first be derivatized with the coupling agent, then the binding species applied to specific regions. The remaining regions may then be blocked or quenched to prevent further binding. The solid phase support may be any material capable of immobilizing the binding species, yet otherwise inert or capable of being rendered inert with respect to the other reagents and components in the assay in which the support is intended to be used. Selection of the support material will depend on the type of species to be immobilized on it as well as the method of immobilization, which will range from adsorption to chemical bonding. In general, any geometrical shape can be used on which one can designate precisely defined areas, preferably discrete, one for each species according to a preselected pattern. In most applications, the support will be a membrane, sheet or strip of material. Examples of such support materials are cellulose nitrate, cellulose and derivatized cellulose, glass fiber, various nylons and derivatives thereof, Teflon and related materials, and composites of these materials as well as combinations of these materials with other materials capable of binding proteins or peptides. Ion exchange resins are examples of the latter. Support materials in the form of membranes or strips are preferred for ease of immersion in sample liquids. The species are applied, through their stock solutions, to the support according to a preselected pattern which is made known to the user as an aid to detection. The pattern consists of precisely defined regions on the support, preferably spaced apart from each other and each containing a single species (or narrow molecular weight range) at a substantially uniform density over the entire area of the region. The size of each region and the overall arrangement are not critical and may vary widely, including for example discrete dots of various sizes, discrete lines of defined length and width and continuous lines of defined width.Parallel lines similar to the bands of an electropherogram are particularly useful, although the present invention enables one to improve considerably over electropherograms by controlling the lines to achieve substantially uniform intensity, sharpness and spacing. The method of application may also vary widely, the optimal method in each case depending on the geometries and arrangement of the binding regions. Application may be achieved, for example, by spray jets or microjets emitted by pressurized nozzles, as well as transfer by capillary action. Application may include controlled spraying of discrete patterns on a stationary membrane surface, controlled spraying of continuous lines or dot patterns on a moving membrane surface, application of measured drops directly to either a stationary or moving membrane surface using a precision micropipetting device, controlled continuous flow application using a wick or brush-type applicator, and any other techniques capable of applying a liquid solution or suspension to a precisely defined area on a solid surface.The method of application as well as the concentration of the stock solution will be selected with a view toward achieving a desired species density on the solid phase support. Densities may vary widely, and optimal amounts will depend on the method of detection contemplated in the assay itself. In most applications, a species density of at least about 10 picograms per square millimeter of support surface area, preferably from about 0.1 to about 10 micrograms per square millimeter, will provide the best results. Once the stock solution or mixture has been applied and the species has been bonded, the support may be processed in a manner similar to a Western blot. This may include a sequence of steps including some or all of washing, blocking and stabilization prior to drying the membrane for packaging. Washing solutions are generally deionized and/or distilled water containing buffer, detergents, bactericidal/bacteristatic agents and proteins such as BSA or BGG. Blocking solutions will generally be aqueous and/or organic solutions containing various proteins and/or detergents. Stabilizing procedures may involve, for example, immersion in or saturation of the bound support with solutions of various proteins or fixative agents such as glutaraldehyde, or lyophilization of the binding species onto the support. The present invention enables one to prepare a multitude of identical solid phase reagents, each bearing the same pattern of binding species at the same densities for performing repetitive assays on a multitude of samples. A particularly useful way of achieving this is by propelling the stock mixtures in jets toward a moving sheet of the support material to form discrete parallel lines of binding regions. The sheet is then cut into strips of substantially equally width in the direction transverse to the parallel lines. The finished reagents which consist of the supports each with an identical pattern of immobilized binding species may be packaged as part of an assay kit containing additional components used with the solid phase reagents in analyzing samples. The nature of the components will depend on the type of assay intended, the assay procedure, and the method by which the presence of binding between species on the support and species in the sample will be detected. A preferred method of detection involves the use of a conjugate of two components--(a) a receptor having affinity for the binding agents in the sample as bound to the species in the solid phase support, and (b) a label capable of providing a detectable signal. Such receptors are generally antibodies specific for antigenic sites common to all of the binding agents. For example, where the binding agents themselves are antibodies of a certain type, such as human IgG, the receptor may be antihuman IgG. Alternatively, other type of immunoglobulins may be used, such as IgM, IgA, IgE, etc. The label may be any of the various types of signal-producing species well known in the biochemical art such as, for example, color-producing species, fluorometric species, and radiometric species.A particularly convenient form is one in which the label component of the conjugate is one component of a two-component signal producing system, the other component being supplied in a separate solution as part of the kit. For example, the component forming part of the conjugate may be an enzyme, while the component in the separate solution may be a substrate on which the enzyme acts. Additional kit components will generally include blocking agents and wash/diluent solutions, optionally containing nonhuman protein. The following is a description of a preparative procedure for the isolation and purification of AIDS viral proteins in accordance with the present invention. This is offered for illustrative purposes only and is intended neither to limit nor define the invention in any manner.

EXAMPLE

AIDS VIRAL PROTEINS AIDS viral proteins of current interest in AIDS antibody assays are those having molecular weights of 160, 120, 65, 55, 41-43, 32, 24, 18 and 15 kilodaltons. The major classes of separation techniques useful for purifying these proteins are affinity, size exclusion, reversed-phase, hydrophobic interaction, ion exchange and hydroxyapatite chromatography. The high performance liquid chromatography (HPLC) mode greatly increases the resolving power of each technique. Affinity chromatography may include any of a number of techniques utilizing immobilized antibodies, lectins, dyes, or other group-specific adsorbents. These media can be specific for one or a number of viral components. To prepare for the separations, the virus is detergent-solubilized using SDS or CHAPS and reduced with mercaptoethanol or dithiothreitol. Preliminary chromatography on a lectin affinity column retains the glycoprotein components of the mixture. More than one type of these adsorbents can be used during the course of a separation, and the type of lectin utilized will depend on the specificity and affinity for the desired viral components. The unretained proteins are then concentrated on a reversed-phase HPLC column, then eluted with an increasing gradient of organic component to separate the remaining proteins. The column in this step may be a bondedphase column such as for example, C-4, C-1, phenyl or cyanopropyl. The glycoproteins retained on the lectin column are eluted and concentrated on a reversed-phase HPLC column as well, from which they are separated by gradient elution. The reversed-phase column in each case concentrates, further separates and desalts the proteins. After reversedphase chromatography, the fractions may be concentrated to dryness in an evaporator such as a Savant Speed-Vac (Savant Instruments, Inc., Farmingdale, New York). The separation may be monitored by removing small samples from each fraction and assaying by Western blot analysis. In an alternative scheme, the first separation may be done by gel permeation chromatography, which separates the high molecular weight proteins (160, 120 and 65 kDa) from each other and from the lower molecular weight components. The lower molecular weight components may then be applied directly to a reversed-phase column for simultaneous desalting and separation. Incomplete separations may then be repeated on other reversed-phase columns. A third alternative is to use ion exchange or hydroxyapatite chromatography in the presence of detergents as the first dimension, followed by a reversedphase final separation. Hydrophobic interaction chromatography in the presence of organic modifiers is a fourth alternative, while affinity chromatography using a dye column such as Affi-Gel Blue is a fifth alternative, both of which would be followed by reversed-phase HPLC. A fourth alternative is the use of an affinity column(s) of immobilized antibodies to one or more of the viral components followed by reversed-phase HPLC. A specific example of an AIDS viral protein separation using a gel permeation technique will now be described. 1. Virus Sample Preparation Purified virus was suspended in a solution of 2% sodium dodecyl sulfate and 700mM 2mercaptoethanol in 60mM TRIS chloride at pH 8.0, and heated for 2 minutes at 100[deg]C. The resulting lysed and solubilized virus was added directly to an HPLC column. 2. Size Exclusion Chromatography The column was a Bio-Rad TSK 250 size exclusion chromatography column, 600 by 7.5 mm (Bio-Rad Laboratories, Inc., Richmond, California), and chromatography was achieved using isocratic elution with a mixture of 20% isopropanol in PBS containing 0.05% Triton X100. The flow rate was 0.75 mi/min and the effluent was monitored at 214 nm. Fractions 0.5 mL in volume were collected by an automatic fraction collector, and the organic component was removed in a Speed-Vac evaporator. The eluate was monitored by Western blot to ensure the separation of the viral proteins. The highest molecular weight viral proteins (160, 120 and 65 kd) were isolated individually by this separation, while the remaining proteins eluted as mixtures--65 and 55 kDa; 55 and 41-43 kDa; 41-43, 30 and 24 kDa; and 24 and 18 kDa. The single component fractions (160, 120 and 65 kd) were desalted on a reversed-phase guard column (C-8, 4.6 by 40 mm) and the solvent evaporated to leave the pure protein. Proteins from the multicomponent fractions were isolated as described in the next sections. 3. Reversed-Phase Hydrophobic Interaction Chromatography The fractions containing 24 kd protein were fractionated on a Bio-Rad TSK Phenyl-5PW column (7.5 by 125 mm) to selectively remove this protein. A linear gradient from 100% solvent A (0.1% aqueous trifluoroacetic acid) to 100% solvent B (0.1% trifluoroacetic acid in acetonitrile containing 10% isopropanol) over thirty minutes was run. The p24 protein passed through the column first while the other proteins eluted subsequently as a mixture. The mixture was further fractionated by reversed-phase chromatography on a C-4 column. The Phenyl-5PW column could also be run in a hydrophobic interaction mode using an inverse salt gradient in the presence of 20% isopropanol with similar results. 4. Further Reversed-Phase Chromatography The protein mixtures from the gel filtration column described in Section 2 above were further fractionated on a Bio-Rad Hi-Pore RP304 column. Prior to fractionation, the samples were evaporated in a Speed-Vac evaporator to remove the organic component, then injected directly onto the column. A linear gradient from buffer A (0.1 % aqueous heptafluorobutyric acid) to buffer B (0.1% heptafluorobutyric acid in acetonitrile) over 60 minutes after an initial 15 minutes at 100% Buffer A was run, at a flow rate of 1 mL/min while monitoring the eluant at 214 nm. Fractions were collected at 0.5 mL intervals and were monitored by Western blot analysis. All remaining protein mixtures were resolved into single components by this method. The foregoing is offered primarily for purposes of illustration. It will be readily apparent to those skilled in the art that numerous additional modifications, variations and substitutions not mentioned herein may also be made without departing from the spirit and scope of the invention.

Claims (24)

1. A method for preparing a solid phase reagent for a biochemical binding assay involving the analysis of a sample for the presence of a plurality of preselected binding agents, said method comprising: (a) isolating from a biological source mixture a plurality of biochemical species each having affinity for one of said binding agents; (b) diluting each of said isolated species in a separate carrier liquid to form a separate liquid mixture of each of said species at a preselected concentration; and (c) applying a preselected quantity of each of said liquid mixtures to a preselected region on a solid phase support to immobilize said species thereon according to a preselected pattern.

2. A method in accordance with claim 1 in which said biological source mixture is a member selected from the group consisting of viral and cell lysates and combinations thereof.

3. A method in accordance with claim 1 in which said biochemical species are a member selected from the group consisting of peptides, polypeptides, proteins and combinations thereof.

4. A method in accordance with claim 1 in which said biochemical species are viral antigens.

5. A method in accordance with claim 1 comprising preparing a plurality of said solid phase reagents by applying in step (c) said liquid mixtures uniformly to each of a plurality of said solid phase supports.

6. A method in accordance with claim 5 in which step (c) comprises applying continuous streams of each of said liquid mixtures in parallel lines along a sheet of said solid phase support, then separating said sheet into strips transverse to said parallel lines.

7. A method in accordance with claim 6 in which said continuous streams are jets emitted from pressurized nozzles.

8. A method in accordance with claim 1 in which step (c) comprises covalently bonding said species to said solid phase support.

9. A method in accordance with claim 1 in which step (c) comprises applying said liquid mixtures to a species density of at least about 10 picograms per square millimeter of surface area of said solid phase support.

10. A method in accordance with claim 1 in which step (c) comprises applying said liquid mixtures to a species density of from about 0.1 to about 10 micrograms per square millimeter of surface area of said solid phase support.

11. A method in accordance with claim 1 in which said carrier liquids of step (b) are buffer solutions.

12. A method for preparing a plurality of solid phase reagents useful in analyzing each of a plurality of biological samples for the presence of a plurality of preselected binding agents, said method comprising: (a) isolating individually from a biological source mixture a plurality of biochemical species each having affinity for one of said binding agents; (b) diluting each of said isolated species in a buffer solution to form a plurality of stock mixtures each containing one of said isolated species at a concentration ranging from about 0.01 to about 10 micrograms per milliliter; (c) propelling said stock mixtures in jets toward a solid phase support sheet moving with respect thereto to bind said isolated species thereto in discrete parallel lines at preselected densities ranging from about 0.1 to about 10 micrograms per square millimeter; (d) blocking unbound regions of said solid phase support to substantially prevent nonspecific binding; and (e) cutting said sheet into strips of substantially equal width transverse to said parallel lines.

13. A method for preparing a plurality of solid phase reagents for use in analyzing each of a plurality of biological samples for antibodies having affinity for preselected antigens of a virus residing in a host cell, said method comprising: (a) lysing said host cell and virus to form a lysate; (b) isolating said preselected antigens individually from said lysate; (c) diluting said isolated antigens in buffer solutions to form a plurality of stock solutions each containing one of said isolated antigens at a preselected concentration; (d) applying preselected quantities of said stock solutions to preselected regions on a solid phase support to bind the antigens therein to said support at preselected densities in a preselected spatial pattern; and (e) blocking unbound regions on said support to substantially prevent nonspecific binding.

14. A solid phase reagent for a biochemical binding assay, comprising a plurality of biochemical binding species bound to a solid phase support in discrete regions thereof at preselected densities, prepared according to the method of claim 1.

15. An assay kit containing a plurality of solid phase reagents each comprising a plurality of biochemical binding species bound to a solid phase support in discrete regions at preselected densities, prepared according to the method of claim 12.

16. An assay kit in accordance with claim 15 further comprising a conjugate of (a) a receptor having affinity for all said binding agents and (b) a label capable of providing a detectable signal.

17. An assay kit in accordance with claim 16 in which said label is one component of a two-component signal producing system, and said assay kit further comprises the remaining component of said two-component signal producing system.

18. An assay kit containing a plurality of solid phase reagents each comprising a plurality of viral antigens bound to a solid phase support in discrete regions at preselected densities, prepared according to the method of claim 13.

19. An assay kit in accordance with claim 18 further comprising a conjugate of (a) a receptor having affinity for antibodies to said viral antigens and (b) a label capable of providing a detectable signal.

20. An assay kit in accordance with claim 19 in which said receptor is anti-human IgG.

21. An assay kit in accordance with claim 19 in which said label is one component of a two-component signal producing system, and said assay kit further comprises a solution of the remaining component of said two-component signal producing system.

22. An assay kit in accordance with claim 19 in which said receptor is anti-human IgG and said label is one component of a twocomponent signal producing system; and said assay kit further comprises a solution of the remaining component of said two-component signal producing system, and a wash solution containing non-human protein.

23. A method as claimed in Claim 1 substantially as herein described with reference to the Example.

24. An assay kit as claimed in Claim 15 substantially as herein described with reference to the Example.