EP0397659A1 - Process for detecting biochemical species and apparatus useful therein - Google Patents

Abstract

The method described serves to detect even reduced quantities of biochemical species, such as specific monoclonal antibodies, while these biochemical species are kept in situ inside a tank containing a culture medium. In a typical embodiment of the present invention, a labeled probe is selectively combined, in the presence of a specific antibody to be detected, with a substrate (20), such as a magnetic substrate, by a bioaffinity technique. The marked probe is then magnetically conveyed to be inspected on a predetermined transparent part of the wall (52) of the tank, where it is inspected. The substrate serves to block any sensitive interference originating either from natural radiation from the culture medium (22) or from a residue of unused material (26) from the labeled probe in the medium.

Description

PROCESS FOR DETECTING BIOCHEMICAL SPECIES AND APPARATUS USEFUL THEREIN BACKGROUND OF THE INVENTION

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. The biological broth is often a culture in which the ligand or antiligand are formed.

An enormous amount of work has been done in the field of detecting specific 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.

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. Sometimes 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. All such procedures, even though they are generally less labor consuming than heterogeneous tests, still require a great deal of manipulation by machine and/or personnel. None of the previously-available homogeneous assays are believed to be as sensitive as the heterogeneous assays known to the art, and few are believed to allow efficient measurement of large molecules, e.g. those above 30,000 daltons. For example, the radial-immunoassay diffusion (RID) assays and "nephleometry" techniques, although capable of measuring large molecules, have undesirably low sensitivities.

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) for 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.

The art stimulating fluorescent light and processing it is well known in analytical chemistry. It is well described in U.S. Patent 4,675,529 to Kushida, in the above-cited Wagner Patent and others of the references cited above. Recently-issued U.S. Patent 4,683,120 shows a centrifugally-assembled "patch" of material, the geometry of which is measured as a criterion of the nature of the composition from which it is assembled. Of course, the above discussion of the background of the innvention is necessarily made with hindsight knowledge of the invention disclosed herein, and it is only with that knowledge that the various references cited atherein could be, or would be, assembled for discussion.

SUMMARY OF THE INVENTION

It is a principal object of the present invention to provide a novel, homogeneous-type process, and novel apparatus useful in such process, for detecting specific analyte-antianalyte reaction products such as ligand - antiligand reaction products or analogous reaction products such as those formed in the reaction between unlabelled DNA segment (e.g. a gene) an complimentary nucleic acid, e.g. RNA segments, which are specific for the DNA segment. Indeed for the purpose _oof this Application, DNA/RNA affinities are generally treated as a species of analyte - antianalyte reaction products.

It is a particular object of the invention to provide in situ means whereby a process of the type described above is substantially free of the need to provide elaborate means to avoid interference by background energy emitted from the biological host composition, i.e. the broth, in which the ligand - antiligand reaction product is formed.

It is a further object of the invention to provide means to carry out such a process, and apparatus for use therein, which is suitably utilized in examining extremely small volumes, even sub-microliter volumes, of biolgical compositions.

It is a further object of the invention to be able to provide a sensitive process, and apparatus for use therein, which allows a very rapid detection of even a very small quantity of a specific antiligand - ligand reaction product even in the presence of a large quantity of potential interfering fluorescence in the composition being subjected to detection or assay.

It is another object of the invention to provide a sensitive homogeneous, and generally applicable, assay for detecting, in addition to small ligands, larger ligands, e.g. those above 5000 daltons, indeed above 30,000 daltons and even hundreds of thousands of daltons. Another object of the invention is to achieve a fast analysis of small quantities of analytes, i.e. materials being subject to detection.

Other objects of the invetion will be obvious to those skilled in the art on their reading of this disclosure. The above objects have been substantially achieved by utilization of a process wherein a substrate means, preferably a material of low translucence such as magnetite- bearing particles, is used to carry a labelled ligand - antiligand product into a segregated, preferably opaque, "patch" of material to be subjected to the detection. In preferred embodiments of the invention, 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", as used herein, includes reflected light from residual labelling dyes, i.e. that would be useful in colorimetric evaluation of a patch, that do not generate radiation as a result of their inherent properties, yet are useful labels in the process of the invention. However, in an appropriate situation, other 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. By "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.

It is to be noted that the term 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.

The term "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.

Some in the art call the material being detected an

"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.

In one advantageous embodiment of the invention, 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. However, 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. However, 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. In such a pre-determined position, the use of a detection means, (e.g. a source of excitation light to impinge on the patch and means for detecting fluorescence from the area) can be easily directed to test for fluorescence from a very small specific patch of the segregated material. 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. In any event, when the carrier bearing a tagged antiligand -ligand of interest is 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.

Thus, not only is the natural background, say fluorescent^"background, of a growing culture medium wholly screened out; but all of the background from the unbound label or tag, say a fluorescence-inducing tag, is wholly screened out by the opaque particulate substrate. The screeing on non-utilized, i.e. unbound tag, is of particular significance because its screening markedly reduces dependence of the value of the detection method on using up most or all of the fluorescent tag before measurement. 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. When the beads to be separated into the patch are to be separated by gravity techniques, it is advantageous that 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. To assure this end, it is desirable that 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. When such compartments are formed in polymer tubing, of the size and type described herein, that 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.

It has been found useful to "block" the surface of the tube which contains reaction compartments with perfluorocarbon liquid coating. A one percent (1%) solution of BSA is also useful in blocking.

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) . Of course, it should be understood that the terms antiligand and ligand are interchangeable and that an antibody can attract an antigen as readily as an antigen can attract an antibody. Thus, the use of all such terms in this Application is relative and as a general rule the "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.

Of course, 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.

Other unexpected advantages of the invention have become apparent. For example, 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.

Another advantage of this detection procedure is that it is not demanding of precise optical lenses or even optical materials, such as glass or quartz, through which the detection may be effective. Indeed, it has been found that light focused, at any suitable angle on the wall of the reservoir selected for detection, is effective. It is most convenient and desirable to measure epifluorescence in many situations. Often, 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.

In the present invention, 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. It should be understood that 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.

It is also to be noted that 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. Moreover, 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. Thus, there is no substantial dedication for test purposes of volume within a reservoir (i.e. compartment) , 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.

Referring to Figure 7, it is seen that 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. In a typical situation, 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). Such a tubing is sold under the tradename Zeus by Zeus Industrial Products, Inc. of Rantan, New Jersey with an inside diameter of about 0.03 - 0.04 inches and a wall thickness of about 0.01 to 0.02 inches. 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. It is very desirable to achieve a quick indication of the presence of cells which secrete, e.g. monoclonal antibodies. Such dectection is enhanced by a small compartment size because the secreting cell will tend to secrete the same number of antibodies as it would in a larger volume. Moreover, the smaller and more spherical shape of the compartment (or shapes, vertically elongated) have more relative surface area per compartment volume for gas excange into the compartment.

This small size is achieved by carefully proportioning the volume of bioculture solution and perfluorocarbon fluid that is pumped into the tube. Thus, it is noted that 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. While 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, as specifically described herein, 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.

It will be understood by those skilled in the art that the measurement of radiation of one type or another from a particle patch is not the only way to utilize the invention. 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.

It will also be understood, that 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. Thus, one material to be detected may be segregated by magnetic technique and another by gravity. Or 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.

All of these procedures should be readily implemented by those people skilled in the biological processing procedures already known in the art, once such people have read this disclosure. For simplicity, the invention is described with reference to the use of a single antiligand, i.e. one useful in detection of a specific monclonal antibody ligand, and of light emanating from a single fluorescent source. However, those skilled in the art will realize that the invention can be utilized in such a way that two or more antibodies can be simultaneously detected by using different atiligands. 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.

Also, it is to be realized that substantial advantages of the process of the invention can still be achieved by removing the particles to be analyzed from the compartment before analysis. For example, after removal the patch may be optically evaluated without a washing step.

Concentration Enhancement

A particularly valuable variant of the process of the invention, one that greatly facilitates early detection of a secreting cell of interest, 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.

In practice, 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.

It is to be emphasized that the cell need not be wholly within the matrix. For example, 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.

It has been found that 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. When a desired antibody is secreted by a cell, 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. Often, they form a "halo" effect because the analyte secretion products (really the ligand analyte-typically a monoclonal) is attached to probes, in the vicinity of the cell, wihtout slowing or obscuring that process by dilution in a larger culture sample. Thus, 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. desired monoclonal secretion products, using this procedure. It should be noted, also, that the secreting cell can be part of the patch. In such a case, the probe can still be included in the patch or it can be part of a liquid composition added after the patch is positioned.

It has been discovered that a good way to achieve this concentration effect is to assure a cell population is in

^"intimate contact with antiligand-treated beads, e.g. magnetic beads. 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. Alternately, the antianalyte-bearing beads might be coated on a reactor surface before the cells are added on the top of the bead.

Also, of course, one can layer the cells on the surface of, say, a well of a 96-well plate and directly on or below a porous patch of beads, e.g. on a clear optical surface.

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. Magnetic Screen

In addition to utilizing opaque beads as substrate, they are also useful as background screeing agents when not coated with an antiligand.

In this instance, antiligand is instead coated on a transparent substrate i.e. microtiter place, film or membrane.

It may, depending on the optical density of a particular test system, be desirable to pull 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.

It is to be understood that the opaque shielding, conveniently carried out with magnetic beads, can be accomplished effectively, using the "prepositioned patches", even after the reaction of the anlyte. In such a situation, 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. In this connection, it is noted that 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. Of course, when all the reactants are in place, 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.

Surprisingly, it is possible to form a layer containing antigen (antianalyte) 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.

In addition to analyzing, or using, the cell surface analysis, it is also possible, using the invetion to analyze intracellular functions. Thus, one may use 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. Among such markers available are enzyme markers, calcium markers, and pH-type markers. Many such markers are available from Calbiochem

Immunochemiσals, Behring Diagnostics, and Division of Hoechst Corporation.

Similarly, cells can be labelled intracelularly 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. In one embodiment of the invention, 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. Of course, the surface need not be that microtiter well. An important aspect of the invention is that the sensitivity of the invention utilizing epifluorescence.

In some embodiments of the invention, related to cells in a pre-placed patch, a group of cell populations can serve as a substrate, e.g. an anlyte substrate. In such applications, an opaque screen can be formed behind the patch, e.g. on the opposite side from the fluorescent light source.. In such an application, say one wherein it is desired to screen a panel of monoclonal antibodies for binding to the surface of tumor cells from a patch, one would use 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. In practice, many samples of the tumor cells would be added into different compartments, say wells of a 96-well microtiter plate.

Then, to each well is added 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. As will be described, 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.

ILLUSTRATIVE EMBODIMENT OF THE INVENTION In this application there is described a preferred embodiment of the invention and suggested various alternatives and modifications thereof, but it is to be understood that these are not intended to be exhaustive and that other changes and modifications can be made within the scope of the invention. These suggestions herein are selected and included for the purposes of illustration in order that others skilled in the art will more fully understand the invention and the principles thereof and will be able to modify it and embody it in a variety of forms, each as may be best suited to the condition of a particular case.

BRIEF DESCRIPTION OF THE DRAWINGS 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.

The following example, in conjunction with Figures l through 6, illustrates an in situ homogeneous assay for detecting the presence of an antibody secreted by hybridoma cells in a tissue-culture media. The tubular arrangement of reactor compartment is similar to that employed by Symthe et al in U.S. Patent 3,491,141. Appropriate sterilization of the tube and perfluorocarbon_. carrier liquid should be taken. EXAMPLE 1

This assay demonstrates the detection of an antibody having a size in excess of 100,000 daltons.

As seen in Figure 1, 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. Bovine serum albumin (BSA) was used to block any further reactive sites on the magnetic- beads. Beads 20 will serve as a substrate for a probe which will bound to the beads and eventually be pulled into an opaque patch. They were dispersed in a tubing-constrained reactor, i.e. compartment 22, which contains a tissue culture medium 24. 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 magnetic-beads 20 through their coatings of goat anti-mouse antibody, which may be called MB/GAM, bind to the newly-secreted antibody 30 (which may be called Ab) as does the "probe" FITC-tagged goat anti-mouse polyclonal antibody 26 (which may be called GAM) . 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.

As seen in Figures 4 through 6, 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, as known in the art, 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. Of course, if there is any magnetic material which has yet to attach to an antibody, it too will be contained in the magnetic patch 42, but it will not contribute to fluorescence.

In a typical operation, 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. For example, 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%. In addition, 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.

Those skilled in the appropriate art will readily be able to select appropriate hardware, i.e. lenses, filters, mirrors, amplifying circuits and signal output devices after reading the description herein above. It is to be particularly noted that background fluorescence which is assignable to unreacted fluorescent probe and the tissue culture itself is wholly shielded from having any substantial effect on the light reflected from patch 42 by the opaque nature of the magnetic patch. The means by which light is made incident on the patch can include any of numerous processes known in the optical art including, but not limited to, thsoe wherein fiber optic means are used for bringing light to the patch and for bringing light from the patch to analysis.

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 C0₂ 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) .

All compartments contained treated magnetic-beads in a typical concentration of about 1.12 x 10 beads per milliliter. The concentration of beads can be much lower. 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 IGG_2a mouse myeloma protein, supplied by ICN Corp., also combines with the magnetic-beads and fluorescent probe and is used as a positive control. It was placed in the other reactors at a concentration of 37.5 nanogram per ml. 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% C0₂ - 95% air environment for 24 hours. Thereupon magnetic-beads were pulled into an opaque patch and the fluorescence oaf the patch was determined.

All reactors from wells containing either secreting cells or IgG_2a were positive for fluorescence. Compartments with non-secreting cells or media only were negative for fluorescence.

The above example uses 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 2

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. When the patch is thin enough to transmit light, 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. For example, 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. Thus, a hybridoma-secreting antibody (IgG-_j_) against human hemoglobin (alpha chain, not beta chain) can be selected using the following strategy. GAM gamma-1 ais 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. Then, 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. a mixture of cells specific for each of the two (2) probes or an antibody with specificity for both probes. To distinguish between these possibilities, in the third case, 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. A typical protocol in which the concentration effect¹ can be observed:

Step One: Then add 21 ml of L45-50² oil to a round bottom Terasaki Plate well. Using sterile technique, add from 3.5-4.0ul of the following reagent solution.

Media - either RPMI 1640 with 20% FCS or Optimum⁴ with 20% FCS. Goat antimouse coated latex magnetic beads 0.7mm diameter 1.95 g/ml density or 1.7mm diameter, 1.07 g/ml density in concentrations from .0010% w/v to 0.003% w/v.

Goat antimouse FITC⁶ - a concentration from 0.375 mg/ml to 1.5 mg/ml. 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.

Anywhere from about 3 to 24 hours after adding the cells, a halo of fluorescence can be seen surrounding those mouse hybridoma cells that are themselves surrounded by GLM BM. Those cells that are not within the pellet of GLM MB will cause the edge of the beads closest to them to become fluorescent Hena, within 3 to 24 hours after adding the cells fluorescence is seen as "Hot Spots" within and around the GLM MB pellet. Furthermore, 24 to 48 hours after adding the cells the "Hot Spots" merge making the entire pallet fluorescence. ι The fluorescence caused by the bi.ndi.ng of mouse anti.body secreted by mouse hybridoma cells that are in or near a pellet of GLM MB and binding of free GLM FITC to the mouse antibody.

9 L45-50 oi.l - si.li.cone oi.l made by Union Carbide

Terasaki Plate - made by Robbins Scientific Optimum - made by GibCo.

Latex magnetic beads - made by Seradyn

Goat and mouse FITC - made by Biomedia

Fluorescence observed through an Olympus 1MT2 fluorescent microscope. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described and all statements of the scope of the invention which might be said to fall therebetween.

Having described our invention, what we now claim is:

Claims

1. A homogeneous process for detecting the presence of a detectably-labelled first antiligand - ligand product which has been formed on a substrate means in a liquid composition contained in a compartment, said process comprising the steps of (a) providing a carrier particle coated with a second antiligand as said substrate means for said detectably- labelled first antiligand - ligand product; (b) collecting at least a portion of said, detectably- labelled antiligand - ligand product borne on said carrier particles from said liquid within said compart- ent in a patch; and (c) measuring the amount of detectably labelled product present in said patch.

2. A process as defined in Claim 1 comprising measuring said patch while it is still in said compartment and utilizng said carrier to shield optical background interference from at least a portion of said composition in said compartment from said patch being evaluated.

3. A process as defined in Claim 2 wherein said evaluation is carried out utilizing an opaque carrier as means to shield optical background interference from said composition in said compartment from said area being detected.

4. A process as defined in Claim 1 wherein said measuring the amount of detectably-labelled product is measured as an inverse function of probe remaining in the liquid composition after it is substantially freed of said substrate-borne antiligand - ligand product by said collecting step.

5. A process as defined in Claim wherein said labelled antiligand - ligand is a labelled nucleic acid-complimentary nucleic acid product.

6. A process as defined in Claim 1 wherein a plurality of different detectable labels are used in forming different probes, each probe bearing antiligands indicative of a different characteristic of said ligand.

7. A process as defined in Claim 1 wherein said particles are magnetic and said patch is opaque.

8. A process as . defined in Claim 1 wherein said evaluation is of an optically-detectable label.

9. A process as defined in Claim 1 wherein said patch is opaque.

10. A process as defined in Claim 1 wherein said detectably-labelled antiligand - ligand product comprises a probe bead having a fluorescent labelling composition within said probe bead.

11. A process as defined in Claims l, 2, 3, 4, 5, 6, or 7 wherein said process is carried out in the presence of live cells.

12. A process as defined in Claim 9 wherein said magnetic particles are collected in said opaque patch by a magnetic field prior to said evaluation step.

13. A process as defined in Claim 1 wherein said collection step is carried out by gravity or centrifugal force.

14. A process as defined in Claims 2, 3, 4, 5 or 6 wherein said collecting is along a translucent area of a wall of said compartment.

15. A process as defined in Cliams 1, 2, 3, 4, 5 or 9 wherein said optically-labelled antiligand - ligand product is labelled with at leas one fluorescece-emitting label.

16. A process as defined in Claims 1, 2, 3, 4, 5 or 9 comprising the additional steps of (a) redispersing said labelled substrate-borne antiligand - ligand material to be detected from said patch and (b) thereafter, recollecting and optically re-evaluating said material for another said optical evaluation.

17. A process as defined in Claims 1 through 10 wherein said ligand - antiligand complex to be detected includes a monoclonal antibody.

18. A process as defined in Claim 12 wherein said ligand - antiligand complex to be detected comprises a monoclonal antibody and wherein the concentration of cells insaid compartment which secreted said antibody is less than 10 cells per 10 microliters of said culture medium and wherein the monoclonal antibody is at a concentration of below 40 nanograms per milliliter.

19. A process as defined in Claim 1 wherein both said first antiligand borne on said substrate and said second antiligand are monclonal antibodies. 21. A process as defined in Claim 1 wherein both said first antiligand borne on said substrate and said second ligand are polyclonal ligands. 22. A process as defined in Claim 1 wherein said first antiligand is polyclonal or monoclonal and siad second antiligand is polyclonal if first antiligand is monoclonal but is monclonal if first antiligand is monoclonal. 23. A process as defined in Claim 1 wherein said compartments are about 1 microliter or less in size and are not substantially elongated in the horizontal direction. 24. A competitive assay of the sample to be tested for the quantitative or qualitative quantity of a known ligand utilizing a probe comprising a known ligand and a label and wherein (1) said probe attaches, through its known-ligand component, to anti-ligand bearing substrate only in a quantity dependent upon the amount of competing quantity of said ligand initially present in the sample being tested; (2) collecting said probe which has attached to said anti-ligand bearing substrate in a patch; and (3) detecting said probe attached to said anti-ligand sub-strate as an inverse measure of the presence and quantity of competing ligand initially present in the sample being tested. 25. An assay utilizing a labelled probe containing a known ligand and a label and wherein (1) said probe formed of a substrated treated with anti-ligand and labelled ligand; (2) adding sample to be tested for the presence of a ligand to said probe; (3) Displacing labelled ligand from the probe-bearing substrate in an amount proportional to the amount of ligand in the sample to be tested; (4) collecting the remaining probe-bearing substrate in a patch; and (5) detecting said probe attached to said anti-ligand as an inverse measure of the presence or quantity of said ligand. 26. Apparatus for detecting an optically-labelled chemical comprising (a) at least one compartment having a transparent wall part; (b) means to concentrate said chemical in a patch within said compartment; (c) said transparent wall of said compartment forming means through which said patch can be viewed; and (d) means to optically evaluate the radiation-emitting or light-reflecting properties of a portion of said small area. 27. Apparatus as defined in Claim 26 wherein said means to concentrate said chemcial comprises a magnet as means to attract magnetic particles, which particles, once attracted, form means to shield background radiation from said means to optically evaluate, and as means to form a substrate for said chemical to be detected, and as means for bearing a fluorescence-emitting label. 28. Apparatus as defined in Claim 26 wherein said compartments have no substantial elongation along the axis of said compartment which is perpendicular to a light path of said detecting apparatus impinging on said compartment wherein gravity is used as means to concentrate said patch at said transparent wall proximate to said means to optically evaluate. 29. A process as defined in Claim 1 wherein said ligand o be detected exceeds 5,000 daltons. 30. A process as defined in Claim 1 wherein said ligand to be detected exceeds 10,000 daltons. 31. A process as defined in Claim 1 wherein said patch is not opaque. 32. A process for analyzing an analyte in a biochemical environment comprising the steps of: (a) immobilizing an antiligand on a substrate; (b) contacting said antiligand with culture media which contains a labelled ligand for said immobilized antiligand; (c) forming a complexed material incorporating said ligand and said antiligand, said material including said ligand and said antiligand bonded to said substrate; and (d) optically evaluating the properties of said complexed material by evaluating the nature of light reflected from or emitted by said material with a light- evaluating mechanism. 33. A process as defined in Claim 32 wherein said substrates are beads and said process comprises the step of forming some said beads into a small opaque patch and carrying out said optical evaluating step on a small protion of said patch; which protion faces away from said culture media and towards said light-evaluating apparatus. 34. A method of facilitating the hogeneous detection of secreting cells in a culture media comprising: (a) forming samples of said media in compartments of less than 10 microliters; (b) incubating said samples for a period of time sufficient for the multiplication of said cells; and (c) detecting the secretion products of said cells in each compartment as an indication of the presence of said secreting cells. 35. A method as defined in Claim 32 wherein said compartmeants acontain less than 1 microliter. 36. A method as defined in Claim 33 wherein said compartments contain less than 1 microliter. 37. A method as defined in Claim 34 wherein said compartments contain less than 1 microliter. 38. A method as defined in Claim 34 wherein said secreted products are antibodies have a size in excess of 100,000 Daltons. 39. A method as defined in Claim 35 wherein said secreted products are antibodies having a size in excess of 100,000 Daltons. 40. A process for detecting an antianlyte-antalyte complex in a culture medium, said complex including, at least, a cell and a probe formed of an antianalyte conjugated to a detectable lable, said cell being subject to detection by labelling the secretion of analyte therefrom with said probe, said process comprising the steps of (a) reducing the quantity of said culture media having access to the cell and any said analyte secretions of said cell by positioning a portion of said cell within a porous matrix, said matrix fromed of immiscible material and prenetrable by said culture medium and by said lable-bearing analyte carried therein; (b) similarly, reducing the mobility of any labelled analyte probe which is within said matrix structure and in proximity to said cell by use of said porous matrix, thereby achieving a concentration of said labelled analyte probe around the cell; and (c) measuring the detectable output of the area of detectable labelled probes which are in the immediate vicinity of said cell, as a criterion of whether or not said cell is secreting said analyte. 41. A process as defined in Claim 40 wherein said cell is adjacent to said surface through which said fluorescent detection is made. 42. A process as defined in Claim 40 wherein said target area is confined around one said secreting cell. 43. Process as defined in Claim 40 wherein said detection is carried out measuring epifluorescence from the target area. 44. A process for detecting an antianalyte-analyte complex conjugated with a fluorescent label in a culture medium, wherein said probe is first placed into the medium with said cells and said allowed time to contact any analyte, causing or allowing any binding product of said probe- to-analyte to form along a transparent surface of a container., and thereupon magnetically attracting some magnetic beads from said medium to provide an opaque shield in a position between any said binding product and said culture medium, from background fluorescence in said medium and then measuring epifluorescnece of light from said target areas as a measure of said binding of said probe to said analyte while said shield is in said position. 45. A process as defined in Claim 44 wherein said probe and analyte bind within the cell. 46. A process as defined in Claim 45 wherein said probe and analyte bind outside the cell. 47. A process for analyzing the epifluorescnece emitted from a target area comprising a fluorescently-tagged- antiligand conjugate by investigating a traget area for said epifluorescnce emitted in response to stimulation by a light source comprising the steps 1. coating and forming said tagged ligand-antiligand on a viewing surface such as the bottom of a transparent plate; 2. drawing an opaque screen of magnetic particles on the opposite side of said target area from said light source; and 3. screening the target area from any background fluorecence on the side of said opaque screen most removed from said light source. 48. A process for detecting a labelled antianalyte- analyte complex wherein at least one of (a) said antianalyte or (b) cells from a culture medium, have been immobilized in a coating immediately adjacent a transparent surface, said process comprising the further steps of 1. reacing said (a) antianalyte and an analyte produced by said cells; and 2. analyzing said reaction product for analyte- antianalyte reaction product by epiemission analysis through said transparent surface. 49. A process as defined in Claim 48 wherein said analysis is by epifluorescence. 50. A process as defined in Claim 48 wherein said patch is opaque. 51. A process as defined in Claim 49 comprising the step of pulling an opaque magnetic patch, to aact as a screen against background fluorescnece before said analyzing step. 52. A process as defined in Claim 1 wherein said first antiligand is polyclonal and said second antiligand is monoclonal. 53. A process as defined in Claim 1 wherein said first antiligand is monoclonal and said second ligand is polyclonal.