Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
  • G01N33/545—Synthetic resin

Definitions

• In vitro diagnostic assays may be used to measure amounts of an analyte found in a bodily fluid sample or tissue sample.
• the analyte must be distinguished from other components found in the sample.
• Analytes may be distinguished from other sample components by reacting the analyte with a specific receptor for that analyte and a labeled reactant or indicator.
• Assays that utilize specific receptors to distinguish and/or quantify analytes are often called specific binding assays.
• the most common receptors include antibodies and other proteins such as Intrinsic Factor, Folate Binding Protein, and Thyroid Binding Globulin. These receptors have a specific binding affinity for an analyte or an analogue of that analyte. Together the binding receptor and analyte are often called a binding pair. Other receptors include DNA or RNA strands. These receptors are useful in detecting the presence of DNA duplexes or DNA:RNA hybrids. Formats for conducting in vitro diagnostic assays include competitive, sequential and sandwich formats. In vitro diagnostic assays, and in particular immunoassays have historically been categorized as being either homogeneous or heterogeneous.
• the entire assay is conducted within a single, essentially fluid phase.
• the reactants, samples and indicators i.e. labeled reactants
• the monitoring of the amount of the indicator is performed without any separation between the receptor bound materials and those materials which remain free in the fluid.
• a physical or chemical signal distinguishes the bound material from the free material.
• the heterogeneous immunoassays there is a physical separation of free material from bound material.
• One component of the binding pair of the immunochemical reaction e.g. an antibody
• the other reactants e.g. analyte and indicator
• the level of indicator is then monitored.
• Homogeneous immunoassays are considered advantageous over heterogeneous assays because they are done entirely in the liquid phase. It has been found that reactions that occur entirely in solution, as in homogeneous assays, proceed quicker than heterogeneous immunoassays.
• the receptor In the homogeneous assay, the receptor is not bound to a stationary solid phase and it is free to move about the solution. Thus, the rate of reaction between the receptor and its binding partner is faster. See, for instance, Newman, D. and Price, C. ; Separation techniques. Principles and Practice of Immunoassay, Macmillan Publisher Ltd. , 1991; pp. 78-95. Additionally, the homogenous assay has fewer steps than the heterogeneous assay, thus is quicker and less expensive to perform than a homogeneous assay. However, homogeneous assays are not as easy to automate as heterogeneous assay.
• the sensitivity of homogeneous assays is lower than the sensitivity of heterogeneous assays.
• Heterogeneous assays are considered advantageous over homogenous assays in that they are more readily adaptable to automation and usually have a higher degree of sensitivity than the homogenous assays.
• solid phase heterogeneous assays have the inherent disadvantage of non-specific binding.
• the receptor when the receptor is bound directly to the solid phase, the movement of the receptor is restricted, and the steric hindrance is increased, thus the rate of the reaction between the receptor and its binding partner can be slow.
• the receptor may be denatured, deactivated or destroyed especially under the harsher conditions of some types of covalent coupling.
• RPIA radial partition immunoassay
• the solid phase of RPIA is an inert glass fiber solid phase support which also serves as a reaction vessel.
• the glass fiber support has low non-specific binding.
• the receptor is immobilized onto a delimited area of the solid phase to form a reaction zone.
• the sample and other reactants, at least one of which is labeled, are added so that they react with the immobilized receptor in the reaction zone.
• Separation of free analyte and/or analyte analogue from bound is accomplished by chromatographic separation on the solid phase support so that the reaction zone is essentially free of unbound materials and the amount of labeled reagent in the reaction zone is monitored.
• the immobilization is accomplished immunologically.
• a primary antibody i.e. antibody to an analyte
• a secondary antibody or antiserum i.e. antibody to the primary antibody
• Immunological immobilization is described in U.S. Patent No. 4,517,288.
• immunological immobilization procedures the essentially soluble immunocomplex is applied to a delimited area of a glass fiber filter paper or other appropriate solid support. The process of immunological immobilization does not require the harsh conditions of some types of covalent coupling.
• Another heterogeneous method which overcomes some of the other disadvantages of homogeneous and heterogeneous assays allows the reactions to occur entirely in solution.
• This method utilizes microparticles as the solid phase. Receptors are immobilized on the microparticles.
• the microparticles are usually latex microparticles and may be paramagnetic.
• the microparticles are added to a solution which also contains the other components required for the immunoassay. After the immunological reaction has occurred, the microparticles carrying the binding pair can be separated from the other reactants and materials, and the free or bound portion can be measured using labeled reactants or indicators.
• microparticles can settle out of solution during the reaction; thus, the benefit of having the particles suspended is lost. Moreover, the particles can settle during storage. Thus, when the particles are pipetted into sample tubes the number of particles dispensed (hence the number of receptors dispensed) may be nonhomogeneous between samples. This can lead to poor experimental precision.
• proteins often exhibit significantly decreased biological activity upon adsorption/conjugation to hydrophobic supports, such as latex, because support-protein interactions can disrupt the three dimensional structure of the protein and steric hindrance is increased.
• the reaction vessel may require shaker flasks or sonicators so that the particles remain suspended.
• additional steps and/or equipment are required thereby increasing the variability and cost of the assay.
• Proteins have been coupled to PEMA to form water- soluble complexes.
• pepsin has been coupled to PEMA and the polymer:protein complex is water soluble upon coupling and the coupled protein retains biological activity.
• pepsin has been coupled to PEMA and the polymer:protein complex is water soluble upon coupling and the coupled protein retains biological activity.
• Ethylene Maleic Anhydride for the Preparation of a Water-soluble Polvanionic Derivative of Pepsin. Preparation and Properties. Acta Chem. Scand. B 28 No. 9; pp. 1098-1100 (1974).
• coupling of proteins to PEMA to form water-soluble complexes has also been described in Shigenori E. et al.. Coupling of the Penicllium
• the present invention involves the use of a polymer as the primary support matrix for a receptor.
• the resulting covalently coupled complex of polymer:receptor is water soluble or suspensible.
• One skilled in the art of immunoassays or DNA hybridization assay would not ordinarily select water soluble receptor bound polymers as a support since separation of the polymer bound material from unbound material is difficult because both are in solution or suspended in solution.
• the separation of water soluble polymer bound reagents by RPIA technology provides a way tq use these polymers.
• the present invention relates to a method of preparing a solid phase support by covalently coupling a specific binding assay receptor or member of a binding pair to a primary support matrix.
• the primary support matrix having the covalently coupled specific binding receptor, is then immobilized on a porous solid phase support.
• the solid phase support serves as a secondary support matrix.
• the binding receptors include, but are not limited to, antigens or fragments thereof, antibodies or fragments thereof, strands of DNA or RNA, proteins, polypeptides or various bio-reactive, non- proteinaceous molecules.
• the primary support matrix is a polymer which has or can be derivatized to have functional groups which can react with the binding receptor.
• the polymer and binding receptors are covalently coupled in a solution. In order for the coupling reaction to occur, the polymer and the receptor must be sufficiently soluble in the coupling solution. Additionally the binding receptor must retain or be capable of recapturing its biological activity. The reaction can be gently stirred with a magnetic stirrer if required.
• the covalently coupled polymer:binding receptor is soluble in aqueous solution.
• the polymers useful in the present invention have a linear backbone and include, but are not limited to, polyethylenemaleic anhydride (PEMA) , polymethyl methacrylate, and polyethyleneimine and mixtures thereof.
• PEMA polyethylenemaleic anhydride
• the polymers can be linear or branched.
• An inert solid support is selected to have low non-specific binding and has interstices or pores within the support that are small enough so the reaction fluid is retained within the support by capillary action.
• the support is advantageously a mat of compressed fibers, such as a mat of glass or synthetic fibers or a porous paper mat.
• the support may, however, be constructed of other porous materials such as sintered glass, ceramics, synthetic spongy material, etc.
• a glass fiber support is preferred because of its inertness. See, U.S. Patent No. 4,517,288.
• the water soluble polymer:binding receptor complex is then applied to the solid phase support to form a specific binding assay complex.
• the specific binding assay complex can be used to determine the presence or concentration of an analyte using assay techniques such as RPIA.
• the water soluble polymer:binding receptor complex can be reacted in solution with the other components required for the reaction to occur.
• the reaction mixture is then applied to a solid phase and the free fraction is separated from the bound fraction.
• this aspect is a means to conduct immunoassays or DNA hybridization assays using polymers as the primary support.
• the use of a water soluble polymer:binding receptor complex allows for the capture of the analyte to occur in solution. Furthermore, the separation of the water soluble polymer bound receptor by RPIA provides a convenient way to separate the water soluble polymer from the sample.
• the polymer functions as the primary support matrix, and a solid inert porous support such as glass fiber, functions as a secondary support. Description of the Drawings Figure 1 shows immobilization of proteins to the water soluble polymer PEMA.
• Figure 2 shows a method for preparing reagents useful in detecting the presence of DNA duplexes or DNA/RNA hybrids.
• a specific binding assay receptor is covalently coupled to a polymer having a linear backbone to form a complex.
• the resulting complex is essentially water soluble or suspensible.
• an effective amount of the complex is added to a delimited area of an inert porous solid support.
• Another aspect of the present invention also is a specific binding assay complex comprising a polymer having a linear backbone and a specific binding assay receptor covalently coupled to the polymer immobilized onto a porous solid phase.
• the binding assay receptor:polymer complex is essentially water soluble.
• the specific binding receptors of the present invention include, but are not limited to, antibodies and fragments thereof and other protein receptors such as Intrinsic Factor, Thyroid Binding Globulin, and Folate Binding Protein, and fragments thereof, antigens, proteins, polypeptides, peptides, DNA and RNA strands.
• the specific binding receptor must retain or be capable of recapturing its biological activity upon coupling with the polymer. It must be sufficiently soluble in the coupling solvent and the final polymer:specific binding reagent must be soluble or suspensible in aqueous solution. Solubilities may be determined from tables or may be determined using routine experimentation.
• the polymers of the present invention have a linear backbone and can be linear or branched.
• the polymers include, but are not limited to, polyethylenemaleic anhydride (PEMA) , polymethyl methacrylate, and polyethyleneimine or combinations thereof.
• PEMA polyethylenemaleic anhydride
• the preferred polymers are at least slightly soluble or suspensible in aqueous solution and the most preferred polymer is PEMA.
• PEMA is slightly soluble in aqueous solution.
• the polymers must be soluble in a solvent that allows the reaction of the polymer and specific binding receptor to occur.
• the preferred coupling solvent is a buffered aqueous solution having a pH favorable to the reaction.
• the coupling reaction can occur by utilizing know coupling reagents such as SIAB.
• the coupling agent is a water soluble carbodiimide.
• the coupling reaction occurs in aqueous solution without the need for additional coupling reagents.
• PEMA reacts with amino groups of the specific binding receptor in aqueous solution at a pH greater than about seven (7) .
• examples of such aqueous solutions include phosphate buffered saline, TRIS, and other biological buffers. See, for example, Friefelder D. , Physical Biochemistry pp. 122-125, W.H. Freeman and Company (1982).
• the reaction of the protein with PEMA is complete when there is no evidence of soluble material. One hour is generally sufficient.
• the reaction mixture will be slightly viscous.
• the water soluble complex can be stored until required.
• the water soluble complex remains soluble over time and is stable.
• Preferably the complex may be stored for at least a year and retain its solubility and reactivity.
• the preparation of the specific binding assay complex may require the transfer of the water soluble covalently coupled binding receptor:polymer complex into a second buffer, hereinafter called a spotting buffer.
• the spotting buffer is selected so that it is non-reactive with the assay components. See, for example, Friefelder D., Physical Biochemistry pp. 122- 125, W.H. Freeman and Company (1982).
• the pH of the buffer may be about 5.0 to 9.0. Physiological pHs are optimum.
• the preferred buffer is TRIS.
• the concentration of the buffer may be about 20-200 mM, preferably 30-
• the spotting buffer can include carrier proteins such as BSA.
• carrier proteins such as BSA.
• the preferred percentage of carrier protein is in the range from about 0 to 4%, most preferably about 0.5%.
• the spotting buffer contains a surfactant, preferably a fluorinated surfactant.
• the preferred surfactant is Zonyl FSN (E.I. DuPont & Co. Cat. No. CH 7152S) .
• the percentage of the surfactant can be preferably from about 0 to 1% and most preferably 0.1%.
• the spotting buffer may be varied to accommodate particular experimental or manufacturing conditions.
• the solid support is selected so that it is inert, thus has low non-specific binding and has interstices or pores within the support are small enough so the reaction fluid is retained within the support by capillary action.
• the support is advantageously a mat of compressed fibers, such as a mat of glass or synthetic fibers or a porous paper mat.
• the support may, however, be constructed of other porous materials such as sintered glass, ceramics, or synthetic spongy material.
• a glass fiber support is preferred because of its inertness. See, U.S. Patent No. 4,517,288.
• the surface of the glass fibers may carry a net charge opposite to the charge of the complex and this may aid in the immobilization.
• the solid phase is assembled from GF/F glass fiber paper (Whatman Inc.) cut into about one inch squares ("tabs") and fit into a snap-fit plastic assembly as described in Giegel et al., Radial Partition Immunoassay. Clin. Chemistry 28:1894-1898 (1982).
• an effective amount of the water soluble or suspensible complex is applied to the solid phase support.
• the effective amounts of complex are those concentrations which yield an effective assay signal and over the range of interest of the analyte.
• the complex would be an antibody to hCG coupled to the polymer.
• Serial dilutions e.g. 1:10, 1:100, 1:1000, 1:10,000 etc
• serial dilutions e.g. 1:10, 1:100, 1:1000, 1:10,000 etc
• Each specific binding assay complex can be evaluated using radial partition immunoassay as disclosed in Giegel et al., Clin Chem. 28:1894-98 (1982) or other methodology.
• Various solutions containing known amounts of the analyte to be detected e.g. calibrators
• the indicator label
• excess reagents are removed from the reaction zone by a wash fluid.
• the amount of label e.g. fluorescence, color, radioactivity
• the dilution which yields an effective signal and range, as demonstrated by the signal output from the calibrators, is selected.
• the desired quantity of specific binding assay complex is prepared using the appropriate dilution.
• the polymer complex is applied to about the center of the tab to form the reaction zone. All subsequent reactions occur at the reaction zone.
• reagent volumes are minimized and separation efficiency is maximized by applying all subsequent reactants to about the center of the reaction zone.
• the specific binding assay complex can be prepared by the end user of the commercial product or by the manufacturer. If the procedure is performed by the manufacturer, the finished tabs are shipped to customers and can be used to analyze the biological material for which the tab is specific.
• the receptor: olymer complexes of this invention once immobilized onto a suitable solid phase, can be used in a wide variety of analytical protocols for analysis of a variety of biological materials.
• the immobilized complex may be useful to assay for analytes from blood, urine or other bodily fluid for the presence of therapeutic drugs, natural or synthetic steroids, hormones, antibodies, DNA strands and other analytes of interest.
• a method for conducting a specific binding assay to determine the presence or concentration of an analyte in a sample comprising: a) covalently coupling a specific binding receptor with specificity for the analyte to a polymer having a linear backbone; b) contacting an effective amount of the covalently coupled specific binding receptor:polymer to a porous solid phase under conditions which effect immobilization of the covalently coupled specific binding receptor:polymer to the porous solid phase; c) applying the sample and an indicator under immunological binding conditions to the porous solid phase; d) determining the amount of indicator immunologically bound to the porous solid phase; and e) correlating the amount of indicator to the presence or concentration of the analyte in the sample.
• the indicator is a labeled analyte, labeled analyte analogue or a labeled second binding receptor such an antibody.
• Substantially all unbound reactants can be removed by washing with an eluting solvent or by other means known in the art.
• the fluid sample and labeled reactant can be applied simultaneously or sequentially.
• the fluid sample and the labeled reactant are added simultaneously and the labeled reactant is a labeled analyte or analogue of the analyte.
• the labeled reactant and any analyte present in the fluid sample compete for a limited number of binding sites on the polymer:binding receptor complex.
• the fluid sample suspected of containing the analyte is reacted with the solid support.
• an excess of labeled analyte or labeled analogue is added.
• the fluid sample and labeled reactant are added sequentially.
• the fluid sample is added before the labeled reactant.
• the labeled reactant is a second binding receptor specific for the analyte at a site different than that of the binding receptor of the complex. The amount of the label is directly proportional to the amount of the analyte.
• the labeled reactant can be labeled directly or indirectly.
• the type of label of the labeled reactant can be, but is not limited to, chromogenic, fluorometric, radiometric, chemometric, luminometric, potentiometric, or enzymatic.
• the polymer complex is applied to a delimited area of the porous solid support, which is most preferably glass fiber, to form the reaction zone. All subsequent reactions, including the wash step, occur in the reaction zone. Where or how the reagents and or samples are introduced is not important. There must be, however, a coincidence of all reactants in a portion of the porous solid support. If the label is enzymatic, the eluting solvent used for the wash step can also contain a substrate specific for the enzyme. Alternatively the substrate can be added in a subsequent step. In a most preferred embodiment, reagent volumes are minimized and separation of bound from unbound is maximized by introducing the reactants at substantially the center of the reaction zone. Volumes of added reagent are dependent upon the size of the solid phase, however volumes less than about 150 ⁇ L are preferred.
• a method for conducting a specific binding assay to determine the presence or concentration of an analyte in a sample comprises: a) providing an effective amount of an aqueous solution of water soluble covalently coupled specific binding receptor:polymer; b) adding the sample under immunological binding conditions to the specific binding receptor:polymer; c) applying an aliquot of the reaction mixture of step b) under immunological conditions to a porous solid phase; d) applying an indicator under immunological binding conditions to the porous solid phase; e) determining the amount of indicator immunologically bound to the porous solid phase; and f) correlating the amount of indicator to the presence or concentration of the analyte in the sample.
• a method for conducting a specific binding assay to determine the presence or concentration of an analyte in a sample comprises: a) providing an effective amount of an aqueous solution of water soluble covalently coupled specific binding receptor: olymer, wherein the polymer has a linear backbone; b) adding the sample and an indicator under immunological binding conditions to the specific binding receptor:polymer; c) applying an aliquot of the reaction mixture of step b) under immunological conditions to a porous solid phase; d) determining the amount of indicator immunologically bound to the porous solid phase; and e) correlating the amount of indicator to the presence or concentration of the analyte in the sample.
• the indicator is a labeled analyte, labeled analyte analogue or a labeled second binding receptor such an antibody.
• Substantially all unbound reactants can be removed by washing with an eluting solvent or by other means known in the art.
• the labeled reactant can be labeled directly or indirectly.
• the label of the labeled reactant can be, but is not limited to, chromogenic, fluorometric, radiometric, chemometric, luminometric, potentiometric, or enzymatic.
• the polymer complex is applied to a delimited area of the porous solid support, which is most preferably glass fiber, to form the reaction zone. All subsequent reactions. including the wash step, occur in the reaction zone. If the label is enzymatic, the eluting solvent used for the wash step can also contain a substrate specific for the enzyme. Alternatively the substrate can be added in a subsequent step.
• the extent to which the indicator is present on the solid phase can be correlated with the amount of unknown analyte as disclosed, for example, in Tijssen, P. , Laboratory Techniques in Biochemistry and Molecular Biology. Practice and Theory of Enzyme
• the polymer complex is applied to about the center of the solid support to form the reaction zone. All subsequent reactions occur in the reaction zone.
• reagent volumes are minimized and separation of the bound fraction from the unbound fraction is maximized by introducing the reactants to substantially the center of the reaction zone. Volumes of added reagent are dependent upon the size of the solid phase. However volumes less than about 150 ⁇ L are preferred.
• the charge of the water soluble or suspensible polymer may be an important factor if the immobilization of the water soluble complex formed is by adsorption. That is, a positively charged complex (e.g. PEMA:HBC antigen complex) may exhibit a significantly greater degree of adsorption to a glass fiber matrix than a negatively charged compound.
• Hydrogen bonding may also play a role in the immobilization of the complex. Additionally, it should be noted that an average molecular weight of the polymer may be important in choosing a water soluble polymer in that the resultant complex should be large enough to remain entrapped in the reaction zone of a solid phase, but not large enough to decrease the efficiency of separation.
• the solid phase that was selected was a paper matrix, Whatman type GF/F glass microfilter paper.
• the solid phase that was selected was a paper matrix, Whatman type GF/F glass microfilter paper.
• the solid phase that was selected was a paper matrix, Whatman type GF/F glass microfilter paper.
• the reaction zone of the tabs containing PEMA-rHBc had a dense, bright concentric spot.
• the tabs containing the PEMA and HBc mixture were fluorescent but the fluorescence was diffuse and brightest at the edges indicating that immobilization had not occurred.
• PEMA-rHBc was diluted 1:93.3 with the buffer described in IIB. Fifty ⁇ L of PEMA-rHBc was admixed with 50 ⁇ L of an HBc-ALP conjugate and 50 ⁇ L of control or patient sample. The mixture was incubated at room temperature for about fifteen minutes. A Stratus Analyzer was used to assay the samples and controls. About 70 ⁇ L of the mixture was added to the center of the reaction zone and incubated at about 37 C for about 5 minutes.
• DNA Hybridization Assay Figure 2 shows a method of preparing reagents useful in detecting the presence of DNA duplexes or DNA:RNA hybrids using 1) DNA immobilized to a water soluble polymer, such as PEMA, and 2) an alkaline phosphatase: T4 32-protein (g32p) conjugate as the enzyme labeled reactant.
• G32p is a phage protein that specifically binds single-stranded DNA.
• DNA:EMA complexes are separated from unbound test DNA by Stratus radial diffusion technology. The signal obtained from the enzyme catalyzed reaction is inversely proportional to the test DNA concentration.
• g32p:alkaline phosphatase serves as the probe for any DNA hybridization assay.
• DNA hybrids could also be detected with biotinylated g32p and an avidin labeled enzyme (e.g. alkaline phosphatase) .

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• 1994
  • 1994-07-06 JP JP7504126A patent/JPH08506902A/ja active Pending
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