EP0933989A1 - Systeme reactif et trousse pour la detection de cellules infectees par le vih - Google Patents

Systeme reactif et trousse pour la detection de cellules infectees par le vih

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Publication number
EP0933989A1
EP0933989A1 EP97910976A EP97910976A EP0933989A1 EP 0933989 A1 EP0933989 A1 EP 0933989A1 EP 97910976 A EP97910976 A EP 97910976A EP 97910976 A EP97910976 A EP 97910976A EP 0933989 A1 EP0933989 A1 EP 0933989A1
Authority
EP
European Patent Office
Prior art keywords
antibody
cell
antigen
mixture
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97910976A
Other languages
German (de)
English (en)
Inventor
Robert A. Hallowitz
Chester F. King
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Tech Imaging Inc
Original Assignee
Bio Tech Imaging Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/732,784 external-priority patent/US5714390A/en
Priority claimed from US08/732,782 external-priority patent/US5817458A/en
Application filed by Bio Tech Imaging Inc filed Critical Bio Tech Imaging Inc
Publication of EP0933989A1 publication Critical patent/EP0933989A1/fr
Withdrawn legal-status Critical Current

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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • 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
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G01N33/54333—Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction

Definitions

  • This invention relates to blood collection and diagnostics. More particularly, the invention relates to blood collection and diagnostics utilizing techniques such as magnetic separation and photodetection.
  • the present invention also relates to methods and an apparatus for detecting the presence of antigens displayed on the surface of cells. More preferably, the present invention relates to the detection of cells infected by human immunodeficiency virus (HIV) and related viruses.
  • HIV-infected cells can be detected and separated from uninfected cells. In a preferred embodiment, separation is achieved by a magnetic field. By coating the infected cells with magnetic particles, transfer of the cells to a precise location is facilitated.
  • a novel aspect of the present invention is a cartridge antigen test which allows for the collection and mixing of blood with reagents in one package, which can be viewed on a fluorescent microscope.
  • the present invention relates to methods and materials useful in the early diagnosis of HIV infections. More particularly, in one embodiment, the invention provides compositions and methods for utilizing commercially available high affinity and highly specific magnetically coupled monoclonal antibodies to an envelope surface glycoprotein of HIV- 1, such as gpl20, along, with commercially available FITC conjugated polyclonal antibodies to the envelope glycoprotein, e.g., gp 120, for the purpose of isolating and fluorescing HIV-1 infected peripheral blood lymphocytes in whole blood.
  • the invention can be used with another invention known as the Mehica GP120 Dectector, an automated fluorescent microscope system that incubates and reads cartridge antigen tests.
  • Infection of a T cell with HIV-1 follows from interaction between an epitope borne by HIV-1 and the CD4 receptor which is located on the T cell surface.
  • the epitope on HIV-1 is borne by the envelope glycoprotein gpl20 (molecular weight 120 kilodaltons).
  • the glycoprotein GP 120 is structurally exposed on the outside of the
  • the gp 120 binds to the CD4 antigens which exist on the cell surface of the helper T cells, etc., and in addition to providing the fusion point between the virus and the T helper cell, gpl20 possesses activity which results in syncytium formation, the mechanism of cell to cell infection with HIV-1, as described in detail in U.S. Patent No. 4,725,699.
  • HIV-1 Human Immunodeficiency Virus Type 1
  • HIV antibody tests have their limitations. Usually antibodies to HIV appear within 3-6 months and as early as 6-8 weeks, but silent infections have been documented in which seroconversion has occurred as late as 3 years from the moment of exposure. Therefore, because an infected person does not develop antibodies immediately, a negative test result cannot rule out HIV infection. It has been shown that the majority (90%) of people first testing positive for
  • HIV will develop AIDS within one year. This strongly suggests that the average person identifying HIV infection has been positive for an average of 8-9 years, in view of the fact that the average interval between infection an AIDS is 9-10 years.
  • a method for determining the concentration of substances in biological fluids wherein magnetically responsive , permeable, solid, water insoluble, micro particles are employed is disclosed in U.S. Pat. No. 4,115,534.
  • Functional magnetic particles formed by dissolving a mucopolysaccharide such as chitosan in acidified aqueous solution containing a mixture of ferrous chloride and ferric chloride is disclosed in U.S. Pat. No. 4,285,819.
  • the micro spheres may be employed to remove dissolved ions from waste aqueous streams by formation of chelates.
  • U.S. Pat. No. 3,933,997 describes a solid phase radio immunoassay for digoxin where anti-digoxin antibodies are coupled to magnetically responsive particles.
  • Small magnetic particles coated with an antibody layer are used in U.S. Pat. No. 3,970,518 to provide large and widely distributed surface area for sorting out and separating select organisms and cells from populations thereof.
  • U.S. Pat. No. 4,018,886 discloses small magnetic particles used to provide large and widely distributed surface area for separating a select protein from a solution to enable detection thereof. The particles are coated with a protein that will interact specifically with the select protein.
  • compositions comprising stable, water insoluble coatings on substrates to which biologically active proteins can be covalently coupled so that the resulting product has the biological properties of the protein and the mechanical properties of the substrate, for example, magnetic properties of a metal support.
  • a diagnostic method employing a mixture of normally separable protein-coated particles is discussed in U.S. Pat. No. 4,1 15,535.
  • Micro spheres of acrolein homopolymers and copolymer with hydrophilic comonomers such as methacrylic acid and/or hyroxyethylmethacrylate are discussed in U.S. Pat. No. 4,413,070.
  • U.S. Pat. No. 4,452,774 discloses magnetic iron-dextran micro spheres which can be covalently bonded to antibodies, enzymes and other biological molecules and used to label and separate cells and other biological particles and molecules by means of a magnetic field.
  • Coated magnetizable micro particles, reversible suspensions thereof, and processes relating thereto are disclosed in U.S. Pat. No. 4,454,234.
  • a method of separating cationic from anionic beads in mixed resin beds employing a ferromagnetic material intricately incorporated with each of the ionic beads is described in U.S. Pat. No. 4,523,996.
  • a magnetic separation method utilizing a colloid of magnetic particles is discussed in U.S. Pat. No. 4,526,681.
  • U.K. Patent Application GB No. 2, 152,664A discloses magnetic assay reagents.
  • Magnetic protein A micro spheres and their use in a method for cell separation are disclosed by Widder,et al. (1979) Clin. Immunol, and Immunopath. 14:395-400.
  • U.S. Patent No. 5,279,936 is a method directed to the separation of a component of interest from other components of a mixture by causing the binding of the component of interest to magnetic particles.
  • the method comprises layering a first liquid medium containing cells and other components with a second medium which is of a different density than and/or different viscosity than the first liquid medium.
  • the cells are bound to paramagnetic particles.
  • the layered first liquid medium and the second liquid medium are subjected to a magnetic field gradient to cause the cell particles to migrate into the second medium.
  • the purpose of isolating the cells in the second liquid medium is to then by a further embodiment to separate the cells from the second liquid medium.
  • U.S. Patent No. 4,935,147 is a method that specifically targets the application of magnetic separation in the assay of organic and inorganic biochemical analytes, particularly those analytes of interest in the analysis of body fluids.
  • the method of the mentioned patent provides a way of separating non-magnetic particles from a medium by virtue of the chemically controlled non-specific reversible binding of such particles to magnetic particles. Because of the small size of the magnetic particles, it also provides for a very rapid binding of a substance to be separated. By then aggregating the particles there is provided a much more rapid and complete magnetic separation than has been achieved by previous methods.
  • this technique of magnetic separation does not apply because of the fact the antigen of interest is bound to cells, and therefore not in solution or in need of agglutinaton for separation.
  • the current invention merely requires the adherence of the many magnetic particles to an infected cell surface to magnetically pull the entire cell of interest to a predetermined point in the reaction vessel for viewing.
  • the prior art collected blood for testing in multiple steps. The first step was to collect the blood into a suitable container from a puncture wound in the skin of a finger or by venipuncture. Then the blood would have to be placed into a container suitable for transporting or mixing with test reagents. Then reagents would have to be added in a multiple step fashion, interrupted by wash steps. The problem with this approach is multiple steps which are time consuming and require training. In the collection of blood, the prior art is still dealing with the lance and test tube methods.
  • the aforementioned U.S. patent 4,777,964 to David Briggs, Kent A. Leger, Brenda Briggs (10/18/88) provides a system for whomever wishes to ascertain whether or not he is carrying the AIDS virus to perform a blood sampling and to forward the sample to a lab for the further testing
  • the kit contains a lance and a tube for collecting the sample and requires the user to seal the tube at the ends with putty.
  • This device and kit is only a means for collecting blood and keeping the sample intact for mailing to a laboratory for further testing. No tests are performed using the appliances provided.
  • the sample must be transferred to a testing vessel and mixed with the appropriate testing medium.
  • the magnetic separator includes a non-magnetic container having a peripheral wall with an internal surface area for receiving the test medium, and magnetic means for generating a magnetic field gradient within the container in which tested material is contained in reaction vessels such as test tubes.
  • reaction vessels such as test tubes.
  • U.S. patent 5,238,810 to Koichi Fujiwara, Juichi Noda, Hiroko Misutani, Hiromichi Mizutani (08/23/93) provides for such a process; however, as with other magnetic separation methods, this method involves multiple apparatus and steps just to collect and prepare the blood samples for testing.
  • This method also focuses on using one reagent for its test, rather than on a double reagent mixture. It provides for various vessel configurations for performing the reaction, but does not include or contemplate a vessel that has served as reagent storage, blood collection, mixture, incubation and viewing device in one.
  • the present invention relates to a variety of assays for detecting and/or separating, e.g., pbls, T-cells, immortalized cell lines, macrophages, artificial liposomes, etc., utilizing magnetic separation technology.
  • it relates to obtaining a blood sample and mixing it with testing reagents in one step, and in one disposable vessel.
  • the vessel can be incubated and the related results of reaction between the reagents and the blood sample can be viewed and read in the vessel by a fluorescent microscope without additional processing for quick and accurate testing.
  • the present invention is directed to blood collection and magnetic separation apparatus and methods in which antibody-coupled magnetic particles and antibody- conjugated flourochromes are used to isolate substances of interest from a nonmagnetic test medium by means of high gradient magnetic separation and identification by application of focused light.
  • An aspect of the present invention relies upon a unique reaction vessel that serves the multiplicity of purposes as stated above.
  • the prior methods of magnetic separation differ in various ways, e.g., because of the incompatibility of reaction vessel configuration with blood sample collection and single-step testing.
  • most magnetic separation devices do not provide for viewing any further reaction within the vessel.
  • the current invention provides a self-contained micro-baggy of reagents that is punctured and permitted to mix with the sample of blood at the same time the sample is being collected.
  • the chamber in which the blood is collected, and in which the reagents are mixed with the blood is also the same chamber or vessel used for incubating the reaction mixture, and further, is the chamber in which magnetic separation of the infected cells, if present, is performed.
  • the Cartridge Antigen Test is a device that permits blood collection, reagent mixing with blood, incubation of the mixture, magnetic separation, and viewing of the test results.
  • the device consists of a well slide with micro-lances, a micro-baggy full of reagents, a mylar cover strip, and a bar code for identification purposes.
  • the present invention also relates to a fluorometric immunoassay in which a pair of manufactured non-competitive antibodies to a surface antigen, such as gpl20, are utilized.
  • a surface antigen such as gpl20
  • One antibody is coupled to paramagnetic particles, while the second in conjugate with FITC.
  • the present invention takes advantage of the technology of immunomagnetic separation developed over the past 15 years to enrich or separate out of a mixture of cells, specific cellular components based on their specific immunological markers. See, e.g., U.S. Patents 4,777,145; 4,731,337; 5,186,827; 5,238,810; 5,279,936; 5,411,863; and 4,935, 147.
  • Fig. 1 is a frontal view of a collection/processing cartridge according to the present invention.
  • Fig. 2 is a side view of the collection/processing cartridge illustrating a well, micro-lances, a micro-baggy and a mylar cover.
  • Fig. 3 is an enlarged view of one of the micro-lances shown in Fig. 2.
  • Fig. 4A-4C are side views illustrating the collection of a blood sample from a test subject.
  • Fig. 5A-5E are side views of the well and illustrating an immunochemical reaction between blood and a two reagent system including incubation, application of a magnetic gradient, and the application of a focused light source on the reagent and blood mixture.
  • Fig. 6A-6C are top and side views respectively, of the cartridge and well illustrating incubation, the application of a magnetic gradient and a focused light source, and the observation of the reaction through a lens.
  • Multi-purpose cartridge and fully automated incubator, magnetic separator and imaging system permit operation by non-medically trained personnel; 3. Appearance of cell-bound gpl20 parallels appearance of viral genetic material, enabling invention to detect HIV presence in same time period as "Gold Standard” PCR at a small fraction of the cost; 4. Functional design of the blood collection/ immunochemistry/ magnetic separation/ imaging cartridge permit complete, self contained, disposable unit that is much easier to handle than "gold standard” PCR test for viral genetic material; 5. Entire test procedure requires minutes to turn around compared with weeks for PCR; 6. Cost per test will be in tens of dollars rather than hundreds. Still further objects and advantages will become apparent from a consideration of the ensuing description and accompanying drawings.
  • a sample of several drops of whole blood is diluted with murine anti-gpl20 monoclonal antibodies coupled to paramagnetic microspheres approximately 0.5 cc Phosphate Buffered Saline (PBS).
  • PBS Phosphate Buffered Saline
  • To the diluled sample is added the Murine anti-gpl20 monoclonal antibodies coupled to paramagnetic microspheres, and the Fluorescein conjugated anti-gpl20 polyclonal antibodies IgG.
  • the sample of diluted whole blood are a small number of HIV infected peripheral blood lymphocytes, bearing CD-4, also bearing numerous exposed gpl20 antigens.
  • both antibodies are non-competitively bound to each and every gp 120 antigen. This renders each HIV infected peripheral blood lymphocytes coated with both the murine anti-gpl20 monoclonal antibodies coupled to paramagnetic micro spheres and the Fluorescein conjugated anti-gpl20 polyclonal antibodies IgG. The uninfected peripheral blood lymphocytes remain uncoated by either of the antibodies.
  • a vessel containing the mixture of incubated blood and reagents can be exposed to a strong magnetic gradient at a predetermined point on the outer surface of the reaction vessel.
  • the magnetic field causes the migration of all HIV infected peripheral blood lymphocytes to the inner surface of the reaction vessel at the maximum point of concentration of the magnetic gradient, thus separating the HIV infected peripheral blood lymphocytes from the uninfected peripheral blood lymphocytes in the diluted whole blood sample.
  • the magnetic separation takes approximately 20 seconds.
  • the predetermined point of maximum magnetic concentration is illuminated by a suitable focused light source at 488 nm wavelength, causing all HIV infected peripheral blood lymphocytes, now aggregated at the at a predetermined point to glow at between 520-540 nm fluorescent light.
  • Fluorescein conjugated anti-gpl20 polyclonal antibodies IgG unbound to HIV infected peripheral blood lymphocytes in the sample of diluted blood, the volume is sufficient and dilution of Fluorescein conjugated anti-gpl20 polyclonal antibodies IgG adequate to provide only a low intensity diffuse background fluorescence as compared to the high intensity of cell bound Fluorescein conjugated anti-gpl20 polyclonal antibodies IgG visible by fluorescence microscopy on the infected cells adhering to the inner surface of the reaction vessel wall.
  • the excess of magnetic particles unbound immunologically to cell surfaces will travel at a much greater velocity to the inner surface of the vessel wall, assuring that before any cell coated with magnetic particles arrive at the vessel wall, there will have formed a dark coating of unbound Murine anti-gpl20 monoclonal antibodies coupled to paramagnetic micro spheres, against which the infected cells will adhere, also providing a nice contrast for the high density of glowing HIV infected peripheral blood lymphocytes.
  • Reagents can be obtained commerically, e.g., Immunodiagnostics, Inc., Murine
  • Anti-gp 120 HIV-1 mAb Coupled to Paramagmetic Microspheres Mnoclonal antibodies of mouse origin can be obtained commerically which are highly specific with high affinity to the gpl20 HIV-1 glycoprotein. They are cross-reactive and cross neutralizing antibodies, which are covalently bonded to Paramagnetic Microspheres. Their coupling ratio is approximately 2.5 micrograms of protein per mg of magnetic microspheres. Specificity testing demonstrates that the Magnetic Murine anti-gp 120 mAb binds recombinant gpl20 (MN, IIIB) peroxidase conjugate as determined by ELISA. The biological activity is defined as the binding of these antibodies to CD-4 bearing, HIV-1 infected cells and HIV-1 infected human peripheral blood lymphocytes. Fluorescein Rabbit Anti-gp 120 HIV- 1 IIIB pAb IgG (e.g., Immunodiagnostics, Inc.)
  • Fluorescein conjugated anti-gp 120 (HIV-1 IIIB) pAg IgG can be highly purified (95% pure) polyclonal IgG before use for FITC conjugation. The conjugate can then be further purified by gel exclusion chromatography. The specificity of this fluorescein conjugated pAb IgG can be defined by its binding to native and recombinant HIV-1 gpl20 in Dot Blot assays and by its staining of cell surfaces in direct immunofluorescence assays. This reagent can be used for direct immunofluorescence assays.
  • This reagent can be used for direct immuno fluorescent staining of cells in the 1 :50 dilution range, while Dot blot assays with purified gpl20 may be performed at a minimum dilution of 1 : 100.
  • Both monoclonal and polyclonal antibodies can be obtained (e.g., see above) which bind to the V3 loop of the HIV-1 envelope glycoprotein gpl20 but which are not competitive, i.e., they attach to different regions of the V3 loop of gpl20. This factor permits them to be used simultaneously for their specific and different purposes.
  • Cell- bound antigen-based test closes the window period created by having to rely on the host immune system to produce antibodies against HIV-1 antigens to around four days; Appearance of cell-bound gpl20 parallels appearance of viral genetic material, enabling invention to detect HIV presence in same time period as "gold standard” PCR at a small fraction of the cost; Entire test procedure requires minutes to turn around compared with weeks for PCR; Increased accuracy and low cost allow it to act as both screening and confirmatory test;
  • This test can also be utilized in an automated format, utilizing a multi-purpose cartridge and fully automated incubator magnetic separator and imaging system, permitting operation by non-medically trained personnel; The test can be contained in a blood collection cartridge to permit complete, self contained, disposable unit that is much easier to handle than "gold standard” PCR test for viral genetic material.
  • the method can be used to test for other viral infections by varying the antibodies combinations, other fluorochromes to could be utilized, the method can be used test for water contamination, the method can be used to separate and identify cancer cells.
  • the present invention relates to an assay for determining the presence of a desired antibody or other binding partner in a test sample.
  • the desired antibody is an antibody generated against an antigen coded for by a virus, e.g., HIV-1, HIV-2, HTLV, HTLV EBV, CMV, SIV, etc.
  • the antibody is a neutralizing antibody or an antibody able to interfere with infection of a cell by the virus.
  • the presence of the antibody in the test sample is determined by its ability to interfere with infection of a target cell by a virus.
  • a target cell is contacted with a virus capable of infecting the cell.
  • the step of contacting can be achieved, e.g., by combining the target cell and virus in a receptacle, such as a test tube, a slide well, a tissue culture flask, etc.
  • a receptacle such as a test tube, a slide well, a tissue culture flask, etc.
  • the step of contacting is performed in a liquid phase; however, a solid phase can also be used.
  • a target cell which is useful in the present invention is one which is susceptible or receptive to being infected by a desired virus, e.g., HIV-1.
  • a desired virus e.g., HIV-1.
  • Various cells can be used, including, primary cells such as CD4(+)T cells or splenic cells, T cell lines, lymphoblastoid cell lines, H9, C8166, Molt, Molt-4, CEM, Jurkat, preferably, CEM74. See, e.g., Virology, 236:208-212, 1997.
  • a target cell can be contacted with the virus under conditions effective to achieve infection of the cell with the virus.
  • factors e.g., cofactors, protein, cytokines, ions
  • environments, ingredients, etc. useful for the virus to infect the cell, e.g., to attach to the cell, enter the cell, and pirate the cell's machinery for its own benefit.
  • These conditions can be routinely determined, e.g., including optimizing pH, temperature, salts, buffers, virus concentrations, cell numbers, etc.
  • the effective conditions also can mean a media or other liquid environment in which invasion of the cell by a virus is accomplished.
  • a media can include growth factors and other compounds which facilitate the virus's entry into a cell.
  • Any suitable buffer system can be used, including, e.g., PBS, Tris, sodium citrate, etc., borate, etc.
  • the combination of the target cell and virus can be referred to as a mixture.
  • the target cell is contacted with the virus, a mixture is formed.
  • the contacting is accomplished in a suitable environment for infection and expression of an antigen associated with viral infection, e.g., gpl20, gp41, etc.
  • a feature of the invention is detection of the viral antigen on the cell surface and detection of agents which interfere with its expression. Detection of the antigen can be achieved directly or indirectly.
  • one or more receptors for a viral antigen e.g., CD4, CKR5, CC-CKR5, CCR5, CKR2, CKR2B, CKR3, CKR4, CXCR4, CCR2, fusin, etc
  • CKR5, CC-CKR5, CCR5, CKR2, CKR2B, CKR3, CKR4, CXCR4, CCR2, fusin, etc can be coupled to a surface (e.g., a magnetic particle, bead, or miscrosphere) and then used to capture cells expressing the viral antigen.
  • a surface e.g., a magnetic particle, bead, or miscrosphere
  • an indirect means of capture is used.
  • a first binding partner specific for an antigen coded for by the virus which is expressed on the cell upon viral infection, is added to the mixture.
  • the term "specific” has its normal art-recognized meaning, e.g., it is has a higher affinity for the viral antigen than other antigens present in the mixture.
  • the binding partner can be added at the same time as the virus, or, after the virus has been added to the mixture.
  • the conditions used are those which permit (e.g., "effective") for the binding partner to attach to the viral antigen as it is expressed on the cell surface, or otherwise displayed by the cell.
  • the binding partner can be any agent which recognizes the viral antigen, e.g., aptamers, PNA structures, peptides, small molecules, antibodies (monoclonal, polyclonal, chimeric, single-chain, divalent, disulfide-stabilized Fv fragments, etc.), receptors for a viral antigen (e.g., CKR5, CD4), etc.
  • Methods for making antibodies are well-known in the art
  • Antibodies can also be obtained from commercial sources, e.g., Immunodiagnostics, Waltham, MA.
  • the cell can be captured directly if the binding partner is attached to a substrate, e.g., a magnetic particle, or, it can be captured by using a second binding partner which is able to specifically recognize the first binding partner, i.e., specifically bind to among a mixture of molecules, antigens, agents, antibodies, etc.
  • a substrate e.g., a magnetic particle
  • the second binding partner is preferably attached to a substrate, e.g., a latex bead, a glass slide, a microwell, a magnetic bead or particle, etc. Attachment of the binding partner to the substrate can be accomplished according to conventional methods. See, e.g., USP 5,543,289; Luk and Lindberg, J. Immunol.
  • a viral receptor as the primary binding partner utilized to capture the target material, e.g., a cell infected with a virus.
  • a receptor for HIV is a preferred reagent for several reasons. It is a universal primary binding site for most subtypes, strains, and clades of the HIV virus and is also on most HIV receptive cells. Because an already HTV-infected cell expresses the gpl20 envelope protein diffusely distributed over its entire membrane surface, and because purified (recombinant or from natural sources) CD4 has a high specificity and affinity for gpl20 , it useful to capture material containing gpl20. For purification, see, e.g., USP 5,603,933; Deen et al., Nature,
  • CD4 can be used indirectly to capture target material, in accordance with the methods described above and below.
  • CD4 can be conjugated to a moiety, e.g., FITC, and employed to capture, e.g., HIV-infected cells in mixture which contains both infected and uninfected cells.
  • Magnetic particles containing anti-FITC antibody can be used in turn to label the cells coated with the CD4-FITC.
  • the mixture can then be passed through a separator column causing positive selection of all the HIV-infected cells in the mixture, while depleting the uninfected cells. After depleting uninfected cells from the mixture, a separation column can be removed from the magnetic filed and the HIV-infected cells eluted with
  • the FITC-conjugated CD4 labeled cells can then be fixed and counted by standard flow cytometry. Cells infected with other viruses can be selected analogously.
  • the second binding partner is attached to a magnetic particle (bead, microsphere, etc.), e.g., as described in USP 5,411,863; USP
  • a magnetic particle can be comprised of any effective type, e.g., ferromagnetic, supermagnetic, paramagnetic, and superparamagnetic.
  • a preferred particle is comprised of iron oxide and polysaccharide.
  • a preferred magnetic bead has a diameter which is less than the diameter of the cell which is to be captured, e.g., about 1-300 nm, about 5-200 nm, about 10-150 nm, preferably, about 20-150 nm, more preferably, about 50-120 nm.
  • the magnetic beads are of a sufficient size that they can form a coating around the cell, e.g., having more than one bead attached to the cell, such as about 10 beads, about 100 beads, about 1000, or about 100-1000 etc. These beads be manufactured or commercially obtained e.g., Milteni Biotech, Germany.
  • the second binding partner is selected for its ability to specifically bind to the first binding partner, i.e., recognize and attach to it with a higher affinity than other components in the cell mixture.
  • the second binding partner can be of any material, e.g., those described for the first binding partner.
  • the first binding partner comprises a moiety which is recognized specifically the second binding particle.
  • the moiety can be attached conventionally to the binding partner.
  • a moiety can be, for instance, a hapten or detectable label, such as a fluorochrome, e.g., FITC, TRITC, R-phycoerythrin, Quantum Red, or Cy3, gold, ferritin, biotin, avidin, streptavidin, green fluorescent protein GFP (Chalfie et al., 1994, Science, 263:802; Cheng et al., 1996, Nature Biotechnology, 14:606; Levy et al., 1996, Nature
  • a fluorochrome e.g., FITC, TRITC, R-phycoerythrin, Quantum Red, or Cy3, gold, ferritin, biotin, avidin, streptavidin, green fluorescent protein GFP
  • a first binding partner can be an anti-gp 120 antibody conjugated to FITC and the second binding partner can be an anti-FITC antibody.
  • the first binding partner can be a receptor for a viral antigen expressed on the cell surface upon viral infection (e.g., CD4, CKR5, fusin, etc).
  • a second binding partner can be selected which is specific for the viral receptor.
  • Such binding partner can be an antibody which recognizes an epitope, etc., on the receptor.
  • the receptor can also comprise a moiety, as mentioned above, and the second binding partner can be an agent which recognizes the moiety, e.g., where the first binding partner is a receptor conjugated to FITC, the second binding partner can be an anti- FITC antibody preferably coupled to one or more paramagnetic microspheres.
  • a second binding partner can be added at the same time as when the virus is contacted with the cell, or it can be added later, e.g., after cell contact, after addition of the first binding partner.
  • a virus or mixture of viruses are added to the cells and then incubated for a sufficient amount of time for the virus to infect the cell and for the cell to display evidence of such infection (e.g., surface expression of gpl20 or gp41).
  • the first and second binding partner can then be added in subsequent and sequential steps. After each addition, optionally, an incubation period is utilized providing adequate time for the binding partner to attach to its substrate. Such times can be routinely determined.
  • a cell-antigen- first binding partner-second binding partner combination is formed.
  • the antigen-first binding partner-second binding partner combination can be referred to as a complex when at least these three components are joined together and attached to a cell.
  • the complex included a magnetic particle, e.g., when the second binding particle is attached to it.
  • separation can be achieved conventionally by a magnetic field. See, e.g., USP Nos. 5,541,072; 5,543,289; 5,238,810; 5,196,827; 4,731,337, e.g., by positive selection.
  • a chamber having an inlet and outlet is filled via the inlet with a sample.
  • the sample contains, e.g., the cells (such as HIV-infected cells) coated with paramagnetic microspheres.
  • a material which is capable of expressing a magnetic field surrounds the filled chamber.
  • a magnetic field is applied to the column, retaining the cells coated with the paramagnetic beads, and allowing the uncoated cells to flow out through the outlet of the chamber.
  • the infected, coated cells can be eluted by releasing the magnetic field.
  • the chamber can comprise any material or matrix, including materials or matriices capable of expressing a magnetic field. Such technology is conventional.
  • USP 5,411,863 describes an apparatus, system, and particles which can be used in the present invention.
  • a related aspect of the present invention is the identification of agents which interfere, modulate, prevent, or enhance, viral infection of a cell.
  • agents can be antibodies, small molecules, aptamers, ribozymes (hammerhead, intron, hairpin, etc.), proteins (cytokines, growth factors, cytokinin antagonists, etc), antiviral agents (proteases, nucleotides, etc.), chemokines, chemokine antagonists (.e.g, antagonists, including antibodies to, e.g., RANTES, MlP-la, MTP-lb).
  • the suspected agent can be added to the mixture as described above and the number of cells captured in the presence or absence of the agent tested or measured.
  • the cells can be pretreated with the agent, e.g., to identify agents which interfere with viral infection after the virus has entered the cell.
  • the agent can be added to the mixture at the same time as the virus, e.g., to identify agents which interfere with viral attachment to the cell or which disable the virus before attachment.
  • Various samples can be used in the present invention, including, any material suspected of containing cells or agents which interfere with viral infection or virus, itself, such as blood, lymph, tissues, organs, in vitro cell culture, urine, saliva, sweat, water samples (e.g., for testing drinking water quality), cell culture media, FBS, serum, feces, food, saline solutions, etc.
  • Such material can be derived from any source or species, including invertebrates, vertebrates, bacteria, mammals, such as humans, apes, monkeys, etc., mollusks, insecta, etc.
  • a related aspect of the present invention involves isolation of viruses from samples, e.g., HIV from plasma.
  • HIV can be isolated from plasma by coupling 2 nm magnetic microbeads with anti-gp4 and/or anti-gp 120.
  • the technique will enrich the virus concentration by immunomagnetic separation with no loss of virus through centrifugation and permit efficient separation from plasma inhibitors of PCR.
  • the same methods described above for cells can therefor can be used for viral isolation.
  • magnetic microbeads e.g., from about 0.5-10 nm, preferably 1-5 nm can be used.
  • Flow cytometry can be accomplished conventionally.
  • the coated cells are eluted from the magnetic separation apparatus. See, e.g., USP 5,411,863. Such cells can then be subjected to flow cytometry according to any method. See, e.g., Hiebert, R.D., "Electronics and Signal Processing", Flow Cytometry and Sorting, Second Ed., Wiley-Liss Inc., pp. 127-155, 1990; M.
  • Test Kit Fig. 1 shows the Cartridge Antigen Test (CAT), comprising a cartridge 16 and a clear rectangular piece of plexiglas, 3/8" thick, 2 wide, and 3" long.
  • the well 14 a 1/4" deep central hemispherical depression in the middle of the cartridge 16, holds the micro-baggy containing the mixture of reagents 12 and three micro-lances 10.
  • the well 14 is covered by a clear mylar strip 18 and adhesive fastener 20.
  • a bar code strip 22 is near the bottom of the cartridge 16.
  • Fig. 2 shows that the well 14 is clear and transparent on the sides, top and bottom, allowing light to pass through the reagent/blood mixture.
  • Fig. 3 shows one of the three micro-lanes 10 which protrude from the bottom of the center of the depression or well 14. Sitting just above the three micro-lances 10 is a micro-baggy containing the mixture of reagents 12.
  • Fig. 4A shows how a test subject holds his/her hand above the well 14 of the cartridge 16.
  • Fig 4B illustrates how the pressing of the thumb on the micro-baggy containing the mixture of reagents 12 above the three micro-lances 10 will cause the test subject to bleed, the blood to be mixed with the reagents.
  • Fig. 4C shows how the cartridge 16 is sealed after collection with the clear mylar strip 18 by lowering the mylar strip 18 into contact with the adhesive strip 20.
  • Fig 5A is a side view of the well 14 before the test subject bursts the micro-baggy containing the mixture of reagents 12.
  • the well 14 contains two reagents needed for the magnetic separation and fluorescent identification: antibodies coupled to paramagnetic microspheres 30 and antibodies coupled with a fluorochrome 32.
  • Fig 5B is a side view of the well 14 covered with the clear mylar strip 18, with the wholl blood sample and reagents prior to incubation.
  • Fig 5C is a side view of the well 14 covered with the clear mylar strip 18, after mixing the whole blood sample with the reagents.
  • Incubation 40 is applied to the cartridge 16 and the uninfected peripheral blood lymphocytes 24 remain unaffected by the reagents.
  • the incubation 40 produces antibodies noncompetively bound to infected peripheral blood lymphocytes 34.
  • Fig. 5D shows the well 14, being exposed to a strong magnetic gradient 42.
  • the magnetic field caused the migration to the inner surface of the well 14 of all the antibodies noncompetively bound to infected peripheral blood lymphocytes 34 to the point of concentration of the magnetic gradient 42, thus separating the antibodies noncompetively bound to infected peripheral blood lymphocytes 34 from the uninfected peripheral blood lymphocytes 24.
  • the magnetic separation takes approximately 20 seconds.
  • Fig. 5E shows a side view of the well 14 after the magnetic separation has occurred.
  • the predetermined point of maximum magnetic concentration is illuminated by a suitable focused light source 44, for example, at 488 nm wavelength, for FICT, causing all antibodies noncompetively bound to infected peripheral blood lymphocytes 34 now aggregated at the predetermined point to glow 48 at between 520-540 nm fluorescent light.
  • the reaction can then be viewed through a microscope or lens of an imaging system.
  • Fig 6 A shows a stop surface view of the cartridge 16.
  • Fig 6B shows the antibodies noncompetively bound to infected blood lymphocytes 34 being separated from the uninfected peripheral blood lymphocytes 24 by the magnetic field to the concentration pont of the magnetic gradient 24.
  • Fig. 6C is a side view of the cartridge 16 and shows how the focused light source 44 is directed through the bottom of the well 14 and the lens 46 placed above the well 14 to view the glow 48 from the reaction.
  • a test subject presses his/her thumb or finger down onto the micro-baggy containing the mixture of reagents 12 on the CAT.
  • the micro-baggy containing the mixture of reagents 12 bursts.
  • the three micro-lances 10 puncture the thumb or finger causing the individual to bleed.
  • the clear mylar strip 18 is pulled down and fastened by adhesive fastener 20, sealing the well 14 containing the blood and the reagents.
  • the first reagent must comprise anti-bodies coupled to paramagnetic microspheres 30 and the second must consist of anti-bodies coupled with a Fluorochrome 32. Both reagents will bind themselves to the infected or target antigen-coated cells during the incubation 40.
  • the mixture in the sealed cartridge 16 is incubated for 3 to 5 minutes at 37 degrees Centigrade.
  • the cartridge 16 is then moved to a viewing platform.
  • a strong magnetic gradient 42 is applied to the side of the well 14. The magnetic field causes the target antibodies, noncompetively bound to infected peripheral blood lymphocytes
  • a forced light source 44 measuring 488 nm is passed through well 14 and the blood and reagent mixture.
  • the focused light source 44 causes antibodies noncompetively bound to infected peripheral blood lymphocytes 34 to glow 48 at the specific emmision frequency determined by the specific fluorochrome.
  • the reaction can be viewed through a lens 46 or predetermined coordinates of the magnetic gradient 42 with the highest concentration at the inner surface of the well 14 where the antibodies noncompetively bound to infected peripheral blood lymphocytes 34 will be located. If there is no glow then the result is negative, and if there is a glow 48 the result is positive.
  • the test subject is identified by the bar code strip 22 attached to the cartridge 16. Accordingly, it can be seen that the invention simplifies the procedures of blood collection, reagent mixing, patient tracking and test reading by unifying all steps into one functional unit.
  • the positioning of the micro-baggy containing the mixture of reagents 12 above the three micro-lanes 10 allows for blood collection and mixing with the reagents in one step.
  • the clear mylar strip 18 is used to cover the exposed well 14 and the cartridge 16 is incubated 40 at 37 degrees Centigrade.
  • the invention works with two reagents.
  • the first reagent consists of antibodies coupled to paramagnetic microspheres 30 so that the infected peripheral blood lymphocytes 26 can be separated from uninfected peripheral blood lymphocytes 24 by applying a magnetic gradient 42.
  • the magnetic field generated by the magnetic gradient 42 will cause the antibodies coupled to paramagnetic microspheres 30 attached to the infected peripheral blood lymphocytes 26 to be drawn to a predetermined location of the interior wall of the well 14.
  • the second reagent consists of antibodies coupled with a fluorochrome 32 so that the infected peripheral blood lymphocytes 26 can be identified if present by applying a focused light source 44 on the well 14 causing the infected peripheral blood lymphocytes 26 to glow at the specific emission frequency determined by the specific fluorochrome.
  • the well 14, covered with a clear mylar strip 18, allows the cartridge 16 to move around and allows the test reaction to be viewed through a lens 46.
  • HIV-1 Isolation System For the numerous instances when it is desirable to separate HIV-1 infected cells from a mixture of uninfected cells, an HIV-1 -infected cell isolation system utilizing imunomagnetic separation can be used. The mixture of infected and uninfected cells is washed, centrifuged and resuspended in PBS . A polyclonal anti-GP 120-FITC is introduced into the resuspended cells and incubated for 10 minutes. The cells are separated by centrifugation and washed. Anti-FITC microbeads are used to separate the fluorescently labeled HIV-1 -infected cells using positive selection columns. Flow cytometry is used to quantitate the separated HIV-1 -infected cells.
  • This same isolation system can be used with other virally-infected cells, such as SIV or HTLV.
  • a neutralization assay is used to determine the quantity of neutralizing antibody activity in sera.
  • a positive control is established by inoculating receptive CM 174 cells (or another receptive viral receptive) in suspension with a mixture of cultured laboratory isolates of HIV-1 (MN and IIIB strains). After 7 days of incubation, the cells are separated by centrifugation, washed and resuspended in PBS.
  • Anti-GP 120-FITC is introduced into the resuspended cells and incubated for 10 minutes. The cells are separated by centrifugation and washed.
  • Anti-FITC Microbeads are used to separate the fluorescently labeled HIV-1 infected cells using positive selection columns. The HIV-infected cells are then quantified using standard flow cytometry.
  • the procedure for determining neutralizing activity of sera is performed by adding serially diluted sera specimens to the mixture of virus and CM174 cells and incubating for the same time as used for the positive control.
  • Anti-GP 120-FITC and Anti-FITC Microbeads are used in the same way as in the positive control to separate and enumerate the HIV-1 -infected cells.
  • the neutralizing activity of each serum specimen is determined by the difference from the positive control in the quantity of HIV- infected cells isolated after treatment and incubation of cells and virus with neutralizing sera.
  • This same assay can be performed with other virallly-infected cells, such as SIV or HTLV-1.
  • an HIV-1 drug screening assay is used to identify new anti-HIV drug candidates' ability to block HIV-1 replication in vitro.
  • a positive control is established by inoculating BTI's receptive CM174 cells in suspension with mixture of cultured laboratory isolates of HIV-1 (MN and IIIB strains). After 7 days of incubation, the cells are separated by centrifugation, washed and resuspended in PBS.
  • Anti-GP 120-FITC is introduced into the resuspended cells and incubated for 10 minutes. The cells are separated by centrifugation and washed. Anti-FITC Microbeads are used to separate the fluorescently labeled HIV-1 -infected cells using positive selection columns. The HIV-infected cells are then quantified using standard flow cytometry.
  • the procedure for determining antiviral activity of new drug candidates is performed by adding serially diluted specimens of the candidate to the mixture of virus and CM 174 cells and incubating for the same time as used for the positive control.
  • Anti-GP 120-FITC and Anti FITC Microbeads are used in the same way as in the positive control to separate and enumerate the HIV-1 -infected cells.
  • the antiviral activity of each candidate is determined by noting the dose related differences from the positive control in the quantity of HIV-infected cells isolated after treatment and incubation of cells and virus with the drug candidate.
  • This same screening assay can be performed using other virally-infected cells, such as SIV or HTLV

Abstract

Cette invention a trait au prélèvement du sang et à des diagnostics. Elle concerne plus particulièrement le prélèvement du sang et des diagnostics reposant sur des techniques telles que la séparation magnétique et la photodétection. Elle concerne, en outre, des procédés et un appareil permettant de déceler la présence d'antigènes se trouvant à la surface de cellules. Cette invention porte, de préférence, sur la détection de cellules infectées par le virus de l'immunodéficience humaine (VIH) ainsi que par des virus apparentés. Il est possible, au titre de cette invention, de déceler des cellules infectées par le VIH et de les séparer des cellules non infectées. Dans un mode de réalisation préféré, cette séparation est réalisé au moyen d'un champ magnétique. Le fait d'enrober les cellules infectées de particules magnétiques facilite leur transfert vers un endroit précis. Un aspect novateur de l'invention repose sur un test d'antigène réalisé à l'aide d'une cartouche et permettant de prélever du sang et de le mélanger à des réactifs dans un conditionnement unique, puis de procéder à l'examen par microscopie en fluorescence.
EP97910976A 1996-10-15 1997-10-15 Systeme reactif et trousse pour la detection de cellules infectees par le vih Withdrawn EP0933989A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US732784 1985-05-10
US08/732,784 US5714390A (en) 1996-10-15 1996-10-15 Cartridge test system for the collection and testing of blood in a single step
US08/732,782 US5817458A (en) 1996-10-15 1996-10-15 Reagent system for detecting HIV-infected peripheral blood lymphocytes in whole blood
US732782 1996-10-15
PCT/US1997/018649 WO1998016101A1 (fr) 1996-10-15 1997-10-15 Systeme reactif et trousse pour la detection de cellules infectees par le vih

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US20010039007A1 (en) * 1996-10-15 2001-11-08 Robert Hallowitz Methods and compositions for determining latent viral load
AU2001271643A1 (en) * 2000-06-30 2002-01-14 Bio-Tech Imaging, Inc. Methods for characterizing the viral infectivity status of host
FR2832507B1 (fr) 2001-11-20 2004-02-20 Stago Diagnostica Methode de detection d'analyte(s) a l'aide de particules magnetiques colloidales
JP4931347B2 (ja) * 2002-10-01 2012-05-16 ファンクショナル・ジェネティクス・インコーポレイテッド 抗−ｔｓｇ１０１抗体およびウイルス感染の処置に対するそれらの用法
WO2006009398A1 (fr) * 2004-07-20 2006-01-26 Cgk Co., Ltd. Système pour la détection d'interactions moléculaires
CN107290534A (zh) * 2017-07-12 2017-10-24 华讯方舟科技有限公司 一种血液细胞捕获芯片及方法

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US4659678A (en) * 1982-09-29 1987-04-21 Serono Diagnostics Limited Immunoassay of antigens
FI844027A (fi) * 1984-10-12 1986-04-13 Labsystems Oy Immunologiskt bestaemningsfoerfarande.
DE68919715T2 (de) * 1988-12-28 1995-04-06 Stefan Miltenyi Verfahren sowie materialien zur hochgraduierten magnetischen abspaltung biologischer materialien.
US5054499A (en) * 1989-03-27 1991-10-08 Swierczek Remi D Disposable skin perforator and blood testing device
US5541072A (en) * 1994-04-18 1996-07-30 Immunivest Corporation Method for magnetic separation featuring magnetic particles in a multi-phase system
US5231993A (en) * 1991-11-20 1993-08-03 Habley Medical Technology Corporation Blood sampler and component tester with guide member

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AU743937B2 (en) 2002-02-07

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