WO1989003533A1 - Procede de detection d'especes biochimiques et appareil utilise dans ledit procede - Google Patents

Procede de detection d'especes biochimiques et appareil utilise dans ledit procede Download PDF

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Publication number
WO1989003533A1
WO1989003533A1 PCT/US1988/003529 US8803529W WO8903533A1 WO 1989003533 A1 WO1989003533 A1 WO 1989003533A1 US 8803529 W US8803529 W US 8803529W WO 8903533 A1 WO8903533 A1 WO 8903533A1
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Prior art keywords
ligand
antiligand
probe
patch
labelled
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PCT/US1988/003529
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English (en)
Inventor
Dennis D. Pietronigro
John L. Sternick
Robert J. Ruemer
Mary Lou Mattes-Pound
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Nygene Corporation
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Publication date
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Publication of WO1989003533A1 publication Critical patent/WO1989003533A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • This invention relates to an improved process and apparatus for carrying out the detection of, and quantitavtive measurement of, bioaffinity chemicals-, e.g. such as those formed by ligand - andtiligand reactions and indicative of the presence of specific antibodies, antigens, cells, cell segments, proteins and enzymes while they are still in a biological broth or other fluid.
  • bioaffinity chemicals- e.g. such as those formed by ligand - andtiligand reactions and indicative of the presence of specific antibodies, antigens, cells, cell segments, proteins and enzymes while they are still in a biological broth or other fluid.
  • the biological broth is often a culture in which the ligand or antiligand are formed.
  • bioaffinitive chemicals e.g. monoclonal antibodies, drugs, receptor-bearing enzyme substrates, or the like.
  • Much of the owrk must rely on radioactive chemicals as indicators. Such tests are called isotopic.
  • the present invention is particularly useful in non-isotopic procedures, but isotopic procedures may be utilized also.
  • heterogeneous assays Such detection processes, or assaying, in the field of immunochemistry has been divided into so-called heterogeneous assays and so-called homogeneous processes.
  • Heterogeneous processes are defined as those wherein it is necessary to separate from the composition being examined, before detection or assay, those assaying reagents which have been selectively bound to the analyte from those which have not been so bound.
  • U.S. Patent 4,652,533 to Jelley discloses such a procedure.
  • the well ELISA (enzyme-linked, immunosorbent assay) and the RIA (radio imuno assay) procedures of the prior art are typical of such heterogeneous assays.
  • the separation of bound entities from unbound entities required in the heterogeneous assays is often facilitated by numerous washing/separation steps.
  • magnetic-beads are used, instead of filters or microtiter plates, as an aid in separating and removing bound species from any composition containing unbound species before carrying out such assay procedures.
  • Homogeneous assays measure bound from unbound entities without the need to separate them from each other, i.e. they both Oan remain in the same compartment while the assay or detection method is carried out.
  • Existing homogeneous tests are not very fast nor sensitive. Few of them are adaptable to in situ procedures, i.e, procedures in which biological entities taking a major part in the process being subject to monitoring are being grown or excreted.
  • U.S. Patent 4,680,275 to Wagner et al discloses a time- delay procedure for avoiding the presence of fluorescence background in a homogeneous test method.
  • Another technique e.g. the invention to which U.S. Patent 4,537,861 to Elings and Nicoli relates
  • a homogeneous non-isotopic immunoassay is the scanning of a spatial pattern which has been created by a plurality of spaced electrodes oar magnets within (or adjacent to) the biochemical composition being assayed. The scanning is carried out in such a way that one can quickly distinguish between a substantially random background fluorescent output and a substantially non-random output associated with the labelled binding reaction which one wishes to detect.
  • DNA/RNA affinities are generally treated as a species of analyte - antianalyte reaction products.
  • a substrate means preferably a material of low translucence such as magnetite- bearing particles
  • a labelled ligand - antiligand product into a segregated, preferably opaque, "patch" of material to be subjected to the detection.
  • the patch shields the zone being subjected to the evaluation from background interference whether its source is unbound tag material or the volume of liquid biological material, or broth, from which the patch has been segregated.
  • Background interference includes reflected light from residual labelling dyes, i.e.
  • tages including isotopic tags can be used.
  • substrate materials like glass can be used, but one must focus any means for receiving light therefrom, so that the background fluorescent is kept out of the optical measurement of e.g. , measurement of fluorescent tag.
  • opaque substrate materials we mean those that can form a substantially opaque patch. Thinner patches are desirable, therefore magnetic particles of limited translucence such as iron oxide-based magnetaic particles are highly advantageous in forming a thin opaque patch. Patches of about a four percent (4%) trans ittance, or less, can be easily achieved.
  • ligand is used broadly herein, in its broader sense of a chemical which has a specific affinity for another chemical which may be termed an antiligand. This definition is generally acceptable in the biochemical art and has replaced the old definition requiring a ligand be a group of atoms around a central metallic ion.
  • liquid is used to describe any material, however viscous, through which the particles bearing ligand and antiligand can migrate into a test patch. Thus, many gels are suitable "liquids" in the practice of the invention.
  • analyte and the material having bioaffinity therefor, an antianalyte.
  • Ligand and antiligand are used herein, but it should be understood that the terms are to be broadly construed to cover such analyte - antianalyte products.
  • this process will utilize magnetic-beads of the type used in the biochemical art to attract a bioaffinitive material, as carrier particle means for forming an opaque patch.
  • carrier particle means for forming an opaque patch.
  • other such particles e.g. those based on carbon- black, barium metal, or other material may be used as relatively opaque carriers.
  • Such carriers are best selected to have uniform chemical and size-distribution characteristics and, of course, to have minimal interference with the biological processes and material being evaluated:
  • Non-magnetic or magnetic particles can be segregated into patches by settling, centrifugal force or even floating if, like some plastics or hollow particles, their buoyancy permits segregation by floating.
  • an advantageous aspect of the magnetic type particles is that they can be caused, by application of a suitable magnetic field, to migrate into a opaque patch, conveniently, but not necessarily, at a pre-determined position adjacent a wall area of the biological compartment being tested.
  • a detection means e.g. a source of excitation light to impinge on the patch and means for detecting fluorescence from the area
  • Another advantage of magnetic particles as a mobile, particuate, substrate material is the relative ease with which they may be redispersed within the bilogical mass being subjected to assay or detection.
  • the carrier bearing a tagged antiligand -ligand of interest when assembled into an opaque patch at the spot of detection adjacent to the wall of the reservoir, or compartment, holding the liquid being assayed, it is found to provide an extraordinarily effective screen against background, or noise, which would be expected to interfere with homogeneous assays carried out on fluids having residual radiation- producing material, e.g. unbound fluorescent probead/or fluorescence interference from biological or other source.
  • the carrier can be a single substrate structure, or a plurality of particles such as beads.
  • a "tag” or “label” herein is any chemical which can be bound to an anti-ligand to form a detectable probe, e.g. a radioactive, reflecting fluorescent or che iluminescent tag.
  • the compartment be so-shaped that beads settling at the bottom of the compartment, ussually the reactor wherein the analyte is being formed, be concentrated in a patch.
  • the compartment not be much elongated along the axis of the tube but, rather, be approximately spherical or even elongated somewhat perpendicular to the axis of the tube.
  • compartments of the bioculture to be tested can be reduced in volume using the perflouorocarbon fluid, preferably about 25 centistokes as obtainable from the Ausimont Group (a subsidiary of Montefluos) as Galden D-25, to less than 1 microliter, e.g. as small as about 0.5 microliters or smaller.
  • the perflouorocarbon fluid preferably about 25 centistokes as obtainable from the Ausimont Group (a subsidiary of Montefluos) as Galden D-25, to less than 1 microliter, e.g. as small as about 0.5 microliters or smaller.
  • a "probe” is typically the antiligand combined with, or coupled to, the detecting agent, e.g. a fluorescent tag. These probes will initially be suspended following the binding of the ligand to be detected to the substrate. The binding of ligand to substrate occurs due to the presence of an anti-ligand (which may be the same or different from the antiligand present in the probe) .
  • an anti-ligand which may be the same or different from the antiligand present in the probe.
  • antiligand and ligand are interchangeable and that an antibody can attract an antigen as readily as an antigen can attract an antibody.
  • antiligand is usually designated as the attractant immobilized on the substrate, and present in the probe, while the ligand is the material in the liquid composition which is selectively attracted to the antiligand.
  • the probe may be formed also with a known ligand attached to label. This is particularly useful in competitive assay. In this case the probe will attach directly to an anti-ligand substrate in an amount dependent on competing ligand in the sample. Also, it is possible to include a substrate in a probe formed of substrate treated with anti-ligand and a labelled ligand which is displaced in an amount proportional to the amount of ligand in the sample.
  • the carrier particles when assembled as an opaque patch provide an extraordinarily effective baseline against which even small quantities of light from specifically-sought, analyte-tagged antianalyte products may be detected, i.e. the complex of substrate, antiligand, ligand and probe.
  • the reservoir will be a liquid "bubble" isolated for evaluation as it moves along as one of a series of such reservoirs inside a plastic tubing, e.g. a fluorocarbon tubing.
  • the term "optically-labelled" is used to describe immunochemical components which are labelled with dyes and, as a consequence of such labelling, can be detected with the use of a suitable light source and detector means.
  • Fluorescence-emitting dyes are very suitable labels and techniques for using them are well known in the art, but other dyestuffs may also be utilized in some situations.
  • the sensitivity and ability to co-exist with living cells of fluorescence-detecting techniques make them preferred techniques for many embodiments of the invention.
  • Appropriate methods of detecting radiation can be used when other tags, e.g. radioactive isotopic tags, are used to form the probes.
  • the labelling can be accomplished in any reasonable way including incorproation of fluorescent material within, e.g., a second set of beads (i.e. probe beads which ultimately can be attracted to the primary carrier beads) glass or plastic beads before the beads are coated with an antiligand, thereby forming a completed probe.
  • a second set of beads i.e. probe beads which ultimately can be attracted to the primary carrier beads
  • glass or plastic beads before the beads are coated with an antiligand, thereby forming a completed probe.
  • the fluorescent tag can be attached to a secondary bead to form a probe bead and can then be treated with an anti-ligand to create a probe.
  • primary glass beads can be used as substate material in conjunction with magnetic beads. Measuring different parameters can be achieved with each kind of bead, e.g. with a different antiligand on each kind of bead.
  • One of the principal advnatages of the invention is that it is carried out with the biochemcial to be detected being measured in situ, i.e. within the medium in which the material being detected is produced.
  • a reservoir i.e. compartment
  • carrying the biological entity to be detected carrying the biological entity to be detected. This factor becomes very important when the volume being analyzed is very small, i.e. in the range of 0.25 to 10 microliters.
  • the detection and assay techniques of the invention can be carried out on small enclosed compartments containing minute volumes of a biolgical composition even as secreting cells are grown and various antibodies or other material such as glycoprotein, or glycolipids or carbohydrates are secreted by the cells.
  • the reservoir, or reactors will be constrained within a polyfluorocarbon tubing (formed of an FEP (fluorine-ethylene polymer) tubing of 18 gauge, i.e.. 0.042 inch inside diameter and 0.012 inch wall thickness).
  • FEP fluorine-ethylene polymer
  • the tubing is sterilizable, non-toxic, and non-wettable by water, and gas permeable.
  • a particular problem is the early detection of specific monoclonal antibodies.
  • the presence of appropriate secreting hybridoma cells is also identified by the existence of a specific antibody in a culture being inspected. Described herein, is a homogeneous assay technique for detecting the presence of a specific monoclonoal antibody by a homogeneous detection of the antibody in the presence of a growing and dividing hybridoma (i.e. in situ) capable of secreting the antibody.
  • small compartment size is achieved by carefully proportioning the volume of bioculture solution and perfluorocarbon fluid that is pumped into the tube.
  • small compartment size properly-shaped, not only facilitates concentration of a patch to be evaluated, but enhances the potential concentration of, e.g., a monoclonal antibody to be detected (as an indication of the presence of a secreting cell) in such compartment, enhancing the detection process.
  • a monoclonal antibody to be detected as an indication of the presence of a secreting cell
  • the invention is specifically described below with respect to an antibody being produced by a secreting hybridoma cell already selected for its ability to express a monclonal antibody, it is to be realized that the invention is also useful in detecting the presence of specific antibodies formed during the process of selecting a hybridoma cell for use in monoclonal production.
  • the process has the extraordinary capability of detecting the presence of less than 10 secreting cells per 10 microliters of culture medium in a compartment of the type described in the illustrative example disclosed herein.
  • Antibody can be detected even in concentrations of 40 nanograms per milliliter and lower.
  • the amount of tag, fluorescent or othe rlabel, that remains in the liquid phase from which the particles have been drawn can also be an inverse measure of the material pulled or settled in the "patch" area. In this process the tag should be substantially different in radiation characteristics from the background.
  • a first analyte to be detected may be associated with a segregated substrate by magnetic or gravity means while another analyte to be detected is segregated by -a different procedure.
  • one material to be detected may be segregated by magnetic technique and another by gravity.
  • a material to be assayed may remain dispersed in bound form in the medium wherein it is assayed, e.g. by an optical measurement, while another material is physically separated by gravity or magnetic technique. This last technique is particularly useful when the tag is not fluorescent or when the background (biologically-generated) fluorescence is very low.
  • each antiligand can be a probe carrying a different label, e.g. different fluorescent tags, which can be distinguishable from one another by optical means - that is by the different wave-length the different labels emit or reflect.
  • a particularly valuable variant of the process of the invention is the use of a "concentration effect" by having the cells themselves and any secretion products of interest constained in a porous network of a solid-phase matrix.
  • the matrix can be formed of opaque antiligand-bearing particles or beads.
  • the primary purpose of such a matrix is to limit mobility and maximize concentrations of the analyte, e.g. and analyte produced by a cell, to be concentrated immediately around the cell where the analyte is labelled with multiple probes. This helps identify the particular secreting cells or the presence of such a cell very quickly.
  • the matrix can have a void volume, i.e. the volume within the matrix filled by liquid.
  • the lower volumes are more preferable in that they tend to provide a greater concentration of secreted products immediately around the cell.
  • the porosity of the patch must be sufficient to allow ligand and probe diffusion therethrough. But the optimum level of porosity will relate to such factors as cell size, analyte size, quanity, and nature of the probe and the like.
  • the cell need not be wholly within the matrix.
  • it could be larger in diameter that the matrix is thick. In such a case, it is preferable to have a major portion of the cell volume within the matrix.
  • a cell itself can be within a substrate (e.g., a pre-formed-patch substrate such as a coated patch which also can contain antiligand-bearing beads and a probe) by painting or any other convenient method prior to reaction with the analyte.
  • a substrate e.g., a pre-formed-patch substrate such as a coated patch which also can contain antiligand-bearing beads and a probe
  • the antibody will be immediately attached to antiligand coated substrate causing fluorescent probe material to subsequently attach, thus making the presence of the cell in the vicinity of the cell most readily detectable.
  • the probe-bearing antibodies will be relatively localized around the cell.
  • the solid substrate matrix is seen to limit the amount of liquid in the zone being inspected and to promote a higher concentration of the anlayte (or a simple cell) and its products with arespect to the liquid, usually a culture medium, in the system.
  • Such a procedure also decreases mobility of the cell and its secretion product and, at the same time, increases the detectability thereof. It is posible to detect a single cell producing, e.g.
  • the secreting cell can be part of the patch.
  • the probe can still be included in the patch or it can be part of a liquid composition added after the patch is positioned.
  • a substrate mix comprising antianlyte- bearing beads and cells to be monitored for analyte production can be coated on a reactor surface, say the interior of a tubular compartment before the remaining culture is fed into the compartment of the bottom of a 96- well microtiter plate or the like.
  • the antianalyte-bearing beads might be coated on a reactor surface before the cells are added on the top of the bead.
  • Another way of forming and utilizing a patch of cells/beads is to place it on a sheet of film, immobilize it with a gel coating if necessary; and, after areas having cells secreting desired antibodies or cells are ientified, cut out and remove the areas as aprt of a cell-harvesting procedure.
  • the sheet is a particularly handy device in that it allows areas bearing the desirable secreting cells to be punched out and recovered easily. All of these procedures provide that the analyte- bearing patch will be immediately adjacent a transparent surface where it can be monitored by light, most favorably epireflective light or epifluorescent light.
  • opaque beads are also useful as background screeing agents when not coated with an antiligand.
  • antiligand is instead coated on a transparent substrate i.e. microtiter place, film or membrane.
  • an opaque patch behind the cell layer or other non-opaque substrate patch after it has been reacted with the probe, thereby shielding the detection system from background radiation.
  • Such an opaque patch is particularly useful in epifluorescent or epireflective systems and has no use at all in light- transmission systems. It is not necessary, in such a reaction, to have the opaque patch-forming particles to be coated with a biochemical.
  • the opaque shielding can be accomplished effectively, using the "prepositioned patches", even after the reaction of the anlyte.
  • the original culture, plus the basic materials used in the invention are placed in the measuring vessel.
  • a secondary population of beads, conveniently magnetic beads other than any beads that may be used in the prepositioned, or "painted", patch is added to the culture overlying the patch.
  • This second population of opaque beads is then pulled (e.g. magnetically or by gravity in an appropriate case in which case they need not be magnetic) in behind the painted- patch, or immobilized substrate, to further shield it from background light emission in the media during measurement of fluorescent, or phosphorescence, or whatever light parameter that is used in the detection process.
  • the above-discussed patient tumor cell investigation procedure is typical of a situation wherein the background may be shielded by magnetic beads after the reaction.
  • the opaque shileding patch may be pulled in behind them before they react. Again, the porosity and diffusion through this shielding patch allows labeling to occur throughout and most importantly on the light stimulating surface of the patch.
  • a layer containing antigen on a substrate, say the substrate of a 96-well plate, cover the antianalyte layer with opaque beads, and only then add over the beads the cell-bearing culture which contains an antiligand-tagged probe.
  • Any anlayte secreted by the cells finds its way through the opaque shielding beads to the antianalyte.
  • the probe may attach to the analyte before, during, or after transit through the shield layer. Note that when opaque beads are used as shields only, it is best to block their surfaces with BSA or the like to avoid undesirable attachment of bioche icals to the beads.
  • an internal cell marker such as the fluorescent markers now available in the art. These can be used with tag materials that will not be detectable until they enter the cell environment. The resulting marker-probe will then indicate quantitatively and qualitatively a specific cell function.
  • markers available are enzyme markers, calcium markers, and pH-type markers. Many such markers are available from Calbiochem
  • cells can be labelled intracellularly with dyes which iteract with DNA and become DNA markers, e.g. ethidium bromide (Calbiochem Immunichemi-cal or DAPI available from Polyscience Inc. of Washington, PA.
  • a microtiter well would be, coated with a series of materials including a culture media under test for an analyte - under the culture media is coated an antiliga'nd bearing layer next to the lower wall of the cell which can be based on beads or some other substrate. Intermediate these two layers is an optical, and very thin layer, or a immobilizing gel such as aga gel.
  • the surface need not be that microtiter well.
  • An important aspect of the invention is that the sensitivity of the invention utilizing epifluorescence.
  • a group of cell populations can serve as a substrate, e.g. an anlyte substrate.
  • an opaque screen can be formed behind the patch, e.g. on the opposite side from the fluorescent light source..
  • the patch's tumor cells as a target, lay them down as the substrate next to the transparent container surface through which detection will take place.
  • many samples of the tumor cells would be added into different compartments, say wells of a 96-well microtiter plate.
  • a different monoclonal antibody each known to correspond to a different cell- surface antigen eptiope. If a monoclonal antibody binds to the patient's tumor cells, this will in turn cause the probe (e.g. fluorescent anti-antibody) to associate with the cell surface. This cell-associated fluorescence may then be measured, preferably by epifluorescence.
  • the optics can be limited to the cell substrate patch by either limiting the depth of focus or by using shielding particles - drawing these behind the cells to form an opaque screen.
  • Figure 1 is a schematic diagram showing a liquid biological culture containing hybridoma cells composition isolated in a compartment within a plastic tubing, best seen in Figure 8.
  • Figure 2 illustrates the same culture as shown in Figure 1 after antibodies are expressed by said cells.
  • Figure 3 is indicative of the same culture after there is substantial binding of both antibodies and a fluorescent probe to magnetic beads.
  • Figures 4, 5 and 6 show the magnetic material pulled into an opaque patch at the side of the compartment.
  • Figure 7 is a schematic view of a light-collecting and measuring system.
  • Figure 8 illustrates, schematically, a typical tube- processing-apparatus for handling a series of biochemical compositions.
  • Figure 9 is a schematic illustration of favored shapes of compartments in which the process of the invention is most favorably carried out.
  • This assay demonstrates the detection of an antibody having a size in excess of 100,000 daltons.
  • polystyrene magnetic-beads 20 of nominal 1.75 micrometer diameter and supplied by the Seragen Company are coated by absorption with goat anti-mouse (GAM) IgG antibody (heavy and light chain type obtained from the Zymed Company) are used.
  • GAM goat anti-mouse
  • BSA Bovine serum albumin
  • compartment 22 Also dispersed in compartment 22 is a so-called tag chemincal, or probe, 26 which is an FITC- tagged goat anti-mouse polyclonal antibody also obtained from Zymed and some preselected hybridoma cells 28 (55.2 hybridoma cells producing IgG 2a ) • Before use, the cells are washed about four (4) times with Hanks balanced-salts solution to remove any free antibody.
  • Other compartments contain either non-secreting 653 mouse myeloma cells as negative control or IgG 2a mouse myeloma protein (a product of ICN Corp.) which is used as a positive antibody control.
  • Figure 2 indicates a new situation wherein, after the passage of time, an antibody 30 is secreted from at least some of the cells 28, i.e. from a secretor cell.
  • the resulting complex is shown in Figure 3 as a chemical composition 32 of the formula, using the terms set forth above: MB/GAM - Ab - FITC/GAM
  • the probe-antibody 26 only associates with beads because of the mouse antibody thereon.
  • a magnet 40 a 7 kilogauss magnet which is about 1 X 1.4 X 7cm in size, is used to pull the magnetic-particle bearing material over to a small area of the perimeter of compartment 22 and thereby to provide a substantially opaque population of such beads in a patch 42 as seen in Figure 6.
  • the quantity of fluorescence emitted in response to a suitably-matched, stimulating light will necessarily be directly related to the quantity of antibody secreted by cells 28 and which attach to both the MB/GAM and the FITC/GAM for form the MB/GAM - Ab - FITC/GAM material.
  • it too will be contained in the magnetic patch 42, but it will not contribute to fluorescence.
  • the fluorescent light used to measure the quantity of labelled product will be generated and measured by a fluorescent microscope comprising a fluorescent light source means to quantitatively measure the fluorescence, i.e. the photomultiplier tube (PMT) and appropriate read out means.
  • the microscope normally is provided with ports to accomodate receptors such as PMTs etc. as described below. It is to be understood that this example relates to the use of FITC.
  • Other light sources are most advantageously used with other fluorescent labels with different optimum excitation frequencies.
  • Other optical systems are also useful in detecting the presence of a probe in the path.
  • an argon laser 46 is used to generate a beam of light 66 (of wavelength 488 nanometers) which is passed through a filter 68 to remove bore light, i.e. incoherent light which is normally emitted by such a laser. Thereupon the resulting beam is passed through a combination 70 of beam expanding and collimating optical lenses to establish a given beam diameter e.g. about 10 microns onto a patch 72, typically of about 100 microns in average diameter which has been collected by magnet 84.
  • the particular size of the beam on the patch is selected to have an intensity (watts/cm") which will avoid excessive irreversible "bleaching" (loss of fluorescence emitting capability) of a fluorescent probe.
  • the beam is reflected at a 45-degree angle off a dichroic beam-splitter 76, and then passes through an objective lens 75 which focuses beam 74.
  • the beam-splitter 76 is selected such that it reflects greater than 90% of argon laser beam 66 and transmits less than 10%.
  • the beam-splitter transmits greater than 90% of the fluorescent spectrum (centered at about 518 nanometers) which is emitted from the patch in response to the fluorescence-stimulating beam 74, and reflects less than 10% of these wavelengths.
  • This fluorescent light 78 emitted from the patch is transmitted through objective lens 75 which collimates the light then passes through beam-splitter 76.
  • Beam 78 carries a substantial amount of reflected light of the stimulating beam 74 so it is filtered through a barrier filter 80 before it is passed through another lens, 81 which focuses the collected light on detector 82 which is conveniently a silicon photodiode.
  • the photodiode output a function of the amount of light in beam 78, is amplified to an output signal.
  • Figure 8 is a schematic view of a typical, sequential, processing procedure utilizing, in general, a known scheme wherein compartment 22 of " about 10 microliters in volue to be subject to analysis are moved through a polytetrafluoreoethylene tubing 52 separated by gas bubbles 50.
  • a liquid material, a perfluorocarbon liquid composition, and sold under the trade designation Fluorinert FC-77 by the 3M Company provides means to facilitate separation and also to facilitate a well-lubricated progressive movement of the reactor along the interior wall of the reaction tube 52.
  • Tubing 52 forms means to allow sufficient nutrient gases and C0 2 to permeate therethrough.
  • the gas or air bubbles between athe compartments can form additional surface for transportation of gases through the Fluorinert carrier phase 60 which surrounds both the gas bubbles and the compartment containing the biological culture.
  • the clinical procedures are as follows:
  • the magnetic-beads, coated and blocked, were added to 96-well, tissue-culture places containing either secreting or non-secreting cells - or, in the case of experimental control, either IgG 2a or the nutrient media (or "tissue culture” media) .
  • the probe is added at a concentration of 375 nanograms of FITC/GAM per ml. From 4 to 250 hybridoma or non-secreting myeloma cells were initially placed in appropriate reaction compartments.
  • the interior of the tubing was lightly coated with a carrier fluid, i.e.
  • Fluorinert FC-77 a composition that is sterilizable, wets the tubing, is gas permeable and must have suitable transparency to enable the optical assay.
  • the reaction mixture was sucked from different wells into the Teflon tube using a manifold and a syringe pumping system, thereby forming reactor compartments within the tube.
  • Sufficient FC-77 was also pumped into the tube so it formed a barrier layer about the compartment of the bioculture, i.e. the reaction mixture.
  • Air was introduced to the tubing, as known in the art, to separate adjacent compartments.
  • the compartments within the tube were then placed in an inclubator at 37°C and in a 5% C0 2 - 95% air environment for 24 hours. Thereupon magnetic-beads were pulled into an opaque patch and the fluorescence oaf the patch was determined.
  • substrate-independent cells There are also substrate-dependent cells, e. ' g. liver cells and fibroblasts. They should be first atached to suitable microcarriers known to the art and sold by the New Brunswick Company.
  • Example 1 The steps of Example 1 are repeated using, instead of magnetic particles, particles relatively high in translucence, i.e. glass beads.
  • the glass beads are segregated into a patch by gravity.
  • care must be taken that the light collected comes exclusively from the patch.
  • the excitation beam and emission beam can be angled somewhat to achieve this result.
  • EXAMPLE 3 It should be realized further that two or more characteristics of a single ligand can be detected by utilizing different probes. Thus, for example, one may wish to establish that an analyte is specific for one antiligand but not for another.
  • the labels can even be of the same general type, e.g. two different fluorescent tags.
  • one probe can be used to detect the fluorescent- spectrum resulting from the anti-ligand specificity for the ligand, another probe can be used to determine the class and isotype of the ligand.
  • a hybridoma-secreting antibody (IgG- j _) against human hemoglobin (alpha chain, not beta chain) can be selected using the following strategy.
  • GAM gamma-1 a is used as a coating on the magnetic bead substrate.
  • Probe 1 is human hemoglobin alpha chain labelled with fluorescent-tag #1, i.e. FITC.
  • Probe 2 is human-he oglobiin, beta chain, labelled with fluorescent-tag #2, e.g. Texas Red.
  • hybridoma cells secreting IgG 1# antibody specific to human hemoglobin alpha chain will be identified in compartments wherein the patch is labelled with tag #1 and no tag #2 - you have presence of a cell population secreting an antibody with the specificity of Probe #1 but not for Probe #2. The reverse is true for cells where the antibody is specific for Probe #2. Compartments in which both labels are detected in the patch signify a third case, i.e.
  • the cells in the compartment are cloned in the same system. (That is the cells are spread out among different compartments (according to the invention) so there is only one cell per compartment.
  • Each compartmnet is re-examined using each of the two probes to determine any compartment having specificity to one probe only.
  • Figure 9 is a schematic view showing such nearly- spherical compartments 91 separated by air bubbles 92 in a plastic tubing 93 with the perfluorocarbon li uind (94) facilitating the separation of the comparments and bubbles, and facilitation movement of the bubbles, and especially compartments along the tube 93.
  • Step One Then add 21 ml of L45-50 2 oil to a round bottom Terasaki Plate well. Using sterile technique, add from 3.5-4.0ul of the following reagent solution.
  • Step Two After a pellet of beads has formed (3 to 24 hours) add 3.5 ml of mouse hybridoma cell solution consisting of RPMI 1640 + 20% FCS or Optimum + 20% FCS with hybridoma cell concentrations of 25 cells per well.
  • Latex magnetic beads - made by Seradyn

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Abstract

Le procédé décrit sert à détecter des quantités même réduites d'espèces biochimiques, telles que des anticorps monoclonaux spécifiques, pendant que ces espèces biochimiques sont maintenues in situ à l'intérieur d'un réservoir contenant un milieu de culture. Dans un mode de réalisation type de la présente invention, une sonde marquée est sélectivement combinée, en présence d'un anticorps spécifique à détecter, à un substrat (20), tel qu'un substrat magnétique, par une technique de bio-affinité. La sonde marquée est ensuite acheminée magnétiquement pour être inspectée sur une partie transparente prédéterminée de la paroi (52) du réservoir, où elle est inspectée. Le substrat sert à bloquer toute interférence sensible provenant soit du rayonnement naturel du milieu de culture (22), soit d'un reste de matière non utilisée (26) de la sonde marquée dans le milieu.
PCT/US1988/003529 1987-10-09 1988-10-07 Procede de detection d'especes biochimiques et appareil utilise dans ledit procede WO1989003533A1 (fr)

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US10725187A 1987-10-09 1987-10-09
US107,251 1987-10-09

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0532359A1 (fr) * 1991-09-13 1993-03-17 Shimadzu Corporation Méthode pour distinguer des cellules
US5374531A (en) * 1993-03-22 1994-12-20 Zynaxis, Inc. Immunoassay for determination of cells
WO2002021130A2 (fr) * 2000-08-21 2002-03-14 Darashkevitch Oleg N Procede et dispositif de detection simultanee de plusieurs composants dans un melange
EP1239284A1 (fr) * 2001-03-08 2002-09-11 The Technology Partnership Public Limited Company Procédé de détection sans séparation et sytème utilisant des particules opaques
US7470540B2 (en) * 2000-10-17 2008-12-30 Febit Ag Method and device for the integrated synthesis and analysis of analytes on a support
US9494581B2 (en) 2004-08-24 2016-11-15 University Of Wyoming System and method for Raman spectroscopy assay using paramagnetic particles

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05249114A (ja) * 1992-03-10 1993-09-28 Nippon Telegr & Teleph Corp <Ntt> 免疫測定法及びその装置
US5601997A (en) 1995-02-03 1997-02-11 Tchao; Ruy Chemotaxis assay procedure
WO2014001982A1 (fr) * 2012-06-29 2014-01-03 Koninklijke Philips N.V. Traitement de particules magnétiques liées et non liées
KR20170099737A (ko) 2016-02-23 2017-09-01 노을 주식회사 접촉식 염색 패치 및 이를 이용하는 염색 방법
US10371610B2 (en) 2016-02-23 2019-08-06 Noul Co., Ltd. Contact-type patch, staining method using the same, and manufacturing method thereof

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US4115535A (en) * 1977-06-22 1978-09-19 General Electric Company Diagnostic method employing a mixture of normally separable protein-coated particles
EP0169434A2 (fr) * 1984-07-26 1986-01-29 Labsystems Oy Méthode pour essai immunologique
WO1986004684A1 (fr) * 1985-02-06 1986-08-14 Labsystems Oy Procede de depistage d'anticorps ou d'antigenes
US4729949A (en) * 1982-05-10 1988-03-08 Bar-Ilan University System and methods for cell selection

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US4649109A (en) * 1984-02-16 1987-03-10 Brandeis University Methods for isolating mutant microorganisms from parental populations

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Publication number Priority date Publication date Assignee Title
US4115535A (en) * 1977-06-22 1978-09-19 General Electric Company Diagnostic method employing a mixture of normally separable protein-coated particles
US4729949A (en) * 1982-05-10 1988-03-08 Bar-Ilan University System and methods for cell selection
EP0169434A2 (fr) * 1984-07-26 1986-01-29 Labsystems Oy Méthode pour essai immunologique
WO1986004684A1 (fr) * 1985-02-06 1986-08-14 Labsystems Oy Procede de depistage d'anticorps ou d'antigenes

Non-Patent Citations (1)

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See also references of EP0397659A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0532359A1 (fr) * 1991-09-13 1993-03-17 Shimadzu Corporation Méthode pour distinguer des cellules
US5374531A (en) * 1993-03-22 1994-12-20 Zynaxis, Inc. Immunoassay for determination of cells
WO2002021130A2 (fr) * 2000-08-21 2002-03-14 Darashkevitch Oleg N Procede et dispositif de detection simultanee de plusieurs composants dans un melange
WO2002021130A3 (fr) * 2000-08-21 2002-10-17 Oleg N Darashkevitch Procede et dispositif de detection simultanee de plusieurs composants dans un melange
US7470540B2 (en) * 2000-10-17 2008-12-30 Febit Ag Method and device for the integrated synthesis and analysis of analytes on a support
EP1239284A1 (fr) * 2001-03-08 2002-09-11 The Technology Partnership Public Limited Company Procédé de détection sans séparation et sytème utilisant des particules opaques
WO2002073198A2 (fr) * 2001-03-08 2002-09-19 The Technology Partnership Plc Essai biologique
WO2002073198A3 (fr) * 2001-03-08 2003-05-30 The Technology Partnership Plc Essai biologique
US9494581B2 (en) 2004-08-24 2016-11-15 University Of Wyoming System and method for Raman spectroscopy assay using paramagnetic particles

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JPH03501884A (ja) 1991-04-25
EP0397659A4 (en) 1991-01-30
EP0397659A1 (fr) 1990-11-22
AU2790889A (en) 1989-05-02
CN1034617A (zh) 1989-08-09

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