JPH03501884A - Method and device for detecting biochemical species - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Background technology of method and device for detecting biochemical species The present invention is formed by, for example, a ligand-antiligand reaction, The presence of specific antibodies and antigens, cells, cell debris, proteins, and enzymes. biocompatible chemicals as shown while in biological processes or other fluids; The present invention relates to an improved method and apparatus for the detection and quantitative tracing of.

The biological broth is the medium in which the ligand or antiligand is formed. It is often a nutrient solution.

Specific biocompatibility, e.g. monoclonal antibodies, drugs, receptor-bearing yeast substrates, etc. A huge amount of research has been conducted in the field of detecting chemical substances. Big Such operations in part rely on radioactive chemicals as indicator agents. Like that This test is called an isotope test. The invention is particularly useful in non-isotopic methods However, isotopic methods are also available.

In the field of immunochemistry, such detection processing and analysis are referred to as so-called heterogeneous analysis. It is divided into so-called homogeneous analysis. Heterogeneous analysis is selective for analytes (analytes) Detection or separation of bound analytical reagents relative to those not so bound Prior to analysis, it is necessary to separate the test element from the compound; is justified. U.S. Patent No. 4,652,533 (inventor: Jelley) discloses such a method. The conventionally well-known ELISA (enzyme-linked immunosolvent analysis) and RIA (radioimmunoassay) methods are typical of such heterogeneous analyses. I'll go.

Separation of bound from non-bound conjugates, which is necessary in heterogeneous analysis, requires numerous washing and separation procedures. It is often made easier by stepping away. Filter or micro-filter the magnetic beads. It is often used as a substitute for titan rice, and is often used as a substitute for titan rice. Separation and removal of bound species from all compounds, including non-bound species, prior to carrying out the analytical method. to help you do this. In homogeneous analysis, when measuring conjugate from unbound conjugate, There is no need to separate them from each other, i.e. both can perform the analysis or detection method. You can stay in the same room while doing so. existing Similar tests are not very fast, nor are they very sensitive. stomach. Some of them are adapted to field work, i.e. monitored processes. Applicable to processes where the growth or release of organisms that predominate in the plant is ongoing. Ru. All such processes generally require less effort than heterogeneous testing, but As such, it requires a large amount of operation by machines and personnel. Previously available homogeneous analyzes This difference is not considered to be as sensitive as the conventionally well-known heterogeneous analysis, and most For example, large molecules of approximately 30,000 Daltons or more can be effectively measured. I can't think of that. For example, radioimmunodiffusion (RID) analysis and “turbidimetric” techniques Although this method can measure large molecules, it suffers from the drawback of low sensitivity.

U.S. Patent No. 4,680,275 (inventor: Wagner et al.) is a similar test method. A time delay method is disclosed to avoid the presence of fluorescent background in the method.

Alternative techniques for allogeneic non-isotopic immunoassays (e.g. Elings and N1col i, U.S. Pat. No. 4,537,861), the organism under analysis formed by multiple spaced electrodes or magnets within (or near) a chemical compound scan the spatial pattern that The above scan shows the label binding reaction that you want to detect. quickly distinguish between the generally disordered output associated with the background fluorescence output and the largely disordered background fluorescence output. It is carried out in such a way that it can be separated.

Techniques for stimulating and processing fluorescence are well known in the field of analytical chemistry. That's it The eel technology is based on U.S. Patent No. 4,675,529 (inventor: Kushida), Fully described in the Wagner patent cited above and other documents cited above. Ru.

Recently issued U.S. Pat. No. 4,683,120 describes centrifugally assembled The quality of the "patch" is shown, and the geometry is such that the patch is assembled from it. It is measured as a property standard of composites.

Of course, the above explanation of the background art is only necessary with hindsight knowledge of the invention described in the present application. The various documents cited therein may be cited above or below for illustrative purposes. It is only due to the knowledge that can be combined.

Summary of the invention The main object of the present invention is to obtain unlabeled pieces of DNA (e.g. genes) and to be formed by a reaction between a corresponding nucleic acid such as a piece of RNA that is identified by Characteristics of ligand/antiligand reaction products and similar reaction products A novel homogeneous method for detecting specific analyte-antianalyte reaction products The object of the present invention is to provide a method and a novel device useful for the method. In fact, the purpose of this application is As a target, DNA/RNA affinities are generally analytes and anti-analytes. Treated as a species of reaction product.

A specific object of the invention is to provide a method of the type described above, in which the ligand antiligand reaction products are released from the biological host compound (i.e. broth) formed therein. Elaborate measures to avoid interference from radiated background energy The object of the present invention is to provide an on-site means that substantially eliminates the need to provide a.

A further object of the invention is a means for carrying out such a method and a method for use therein. devices in which the volume of biological compounds is very small, and in some cases ultra-microscopic. Provide suitable means and equipment for testing even liters. It is in.

Furthermore, the present invention provides a highly sensitive method and apparatus for use in specific anti-reflection methods. The amount of Gund-ligand reaction products is very small, and the amount of Gand/Ligand reaction products is very small and cannot be detected or analyzed. Even when there may be a large amount of interfering fluorescence in the compound, The object of the present invention is to provide a method and apparatus that can quickly perform the following steps.

Furthermore, it is an object of the present invention that in addition to small ligands, e.g. In some cases, thick rigs over 30,000 Daltons, and even over several hundred thousand Daltons. To provide highly sensitive and versatile homogeneous analysis technology for detecting Ru.

Another object of the invention is to provide fast analysis of small amounts of analyte (i.e. the substance being detected). We are in the process of doing this.

Other objects of the invention will become apparent from the description below.

According to the method utilized to achieve the above-mentioned objective, the substrate means (preferably magnetic iron) (low-transparency materials such as steel particles) to detect portions of the target that are preferably opaque. Delivery of labeled Quilligant antiligand products into separated material “patches” . In a preferred embodiment of the invention, the punch covers the area under evaluation from background interference. This shielding occurs when the source is the unbound tag material or This is done whether the patch is a separated liquid biological substance or a broth. child The term "background interference" used here refers to residual labeling, which includes reflected light from the dye. i.e., it is useful for the photometric evaluation of the patch, as a result of its inherent properties Residual labels that do not generate radiation and are useful in the method of the present invention Includes reflected light from the dye. However, in appropriate circumstances, other Tags can also be used. A substrate material such as glass can be used, but it cannot receive light from it. Focus on any measures taken to ensure that background fluorescence is measured, e.g. fluorescent tags. optical measurements such as

An "opaque" matrix material is one that is capable of forming a substantially opaque patch. Ru. The thinner the patch, the better; therefore, the transparency of iron oxide-based magnetic particles etc. is limited. magnetic particles are particularly effective in forming thin opaque patches. Approximately 4% Patches with the following transparency can be easily realized.

The term "ligand" is used in a broad sense in the field of chemistry, and is used to refer to antiligands. Here, we refer to a substance that has a specific affinity for another chemical substance, which can be called I am using it. This limitation is generally accepted in biochemical technology and is The old restriction that the Gund is a group of atoms surrounding a central metal ion has already been met. It's all about what's being replaced.

The term "liquid" refers to a viscous substance through which the ligand and antiligand as representing any material that allows particles with a I am using it. That is, many gels are suitable for use in practicing the present invention. Chill with your body.

In some conventional techniques, the substance to be detected is called an ``analyte'', and the Substances that have physical affinity are called anti-analytes. Here, the ligand and a Although anti-ligands are used, they are shall be broadly construed to cover light products.

In one embodiment of the invention, the method uses biochemical techniques to attract biologically compatible substances. A carrier for forming opaque patches using magnetic beads in a format such as those used in Use it as a particle means. However, for example, carbon, metal beryllum, or other substances Particles based on can also be used as relatively opaque carriers. That's it The carrier has uniform chemical and dimensional distribution properties and is of course compatible with the biology being evaluated. They are best selected to minimize interference with commercial methods and materials. Non-Sun or Magnetic Separation of air particles within the patch can be done by centrifugal force, and in some cases In some cases, such as some plastics or hollow particles, which can float and separate due to their buoyancy. If so, you can also use it. However, the convenient form of the miss-form particles can be moved into the cine transparent patch by applying an appropriate magnetic field. and, in that case, it is not necessary, but close to the wall area of the biological chamber under test. This means that it can be conveniently moved to a predetermined position. in place like that , detection means (e.g., a source of excitation light applied to the patch and detection of fluorescence from that range) (means to separate) allows fluorescence from very small specific patches of separated substances Tests can be easily carried out. Magnetism as a mobile particulate matrix material Another advantage of using air particles is that they can be Second, it is relatively easy to redistribute them. Either However, if the carrier holding the tagged antiligand/ligand of interest is , an opaque patch at the detection spot adjacent to the wall of the container or chamber containing the liquid under analysis. When incorporated into a screen, it is extremely effective against background or noise. It is known that a horn is formed.

The background or noise may be, for example, non-conductive from biological or other sources. The combination is carried out in a liquid with a residual radiation emitting substance that is a fluorescent probede or a fluorescent interference. This is thought to interfere with homogeneous analysis. The carrier can be a single substrate structure. Alternatively, it may be a plurality of particles such as beads.

i.e. the natural bank ground of the growing culture medium, i.e. the fluorescent background. Not only are the lands completely shielded, but also unbound labels or tags, i.e. However, all background from fluorescence-inducing tags is also completely absorbed by the opaque particle matrix. Physically shielded. Shielding unused (unbound) tags is especially important and why is used for shielding and for depleting most or all of the fluorescent tag before measurement. This is to significantly reduce the dependence of the value of the detection method used. “Tap” here. ``label'' or ``label'' refers to, for example, a radioactive reflective fluorescent or chemiluminescent sticker. can bind to antiligands to form detectable probes such as

If the beads to be separated into patches are separated by gravity techniques, the shape of the chamber is The beads remain at the bottom of the reactor (usually the reactor in which the analyte is formed). It is convenient to set it to focus on patches. To ensure this, the room should not be very long along the axis of the tube and should be roughly spherical, or in some cases Therefore, it is desirable to have a shape that extends somewhat perpendicularly along the axis of the tube. Yes.

Such a chamber may be formed in a polymer tube of dimensions and form as described herein. The chamber of the biological culture to be tested is then preferably approximately 25 centistokes thick. , D- from Ausimont Group (a subsidiary of Montefluos) The volume of perfluorocarbon liquid used as obtained as 25 decrease to less than a chlorliter (e.g., about 0.5 microliters or less) can be done.

By “blocking” the surface of the tube surrounding the reaction chamber with a perfluorocarbon liquid coating, It has been found to be beneficial. A 1% solution of BSA also helps with occlusion.

The “probe” is typically combined with a detection agent, e.g. a fluorescent tag. or a linked antiligand. These probes are initially in suspension followed by binding of the ligand to be detected to the substrate. Ligand to substrate The binding of is caused by the presence of an antiligand (the antiligand is attached to the probe). may be the same or different from the antiligand present). Of course, antiligands and The terms ``antigen'' and ``ligand'' are interchangeable; can attract antigens as easily as it can. In other words, the present application All such terms are used relatively and as a general rule: An “antiligand” is an attractant that is usually immobilized in the substrate and present in the probe. -10,000, the ligand is a liquid that is selectively attracted to the antiligand. It is made up of substances in the body's compounds.

Of course, the probe can also be formed with a known glue gunt attached to a label. this is a competition Particularly useful in analysis. In this case, the probe is the competing ligand of the sample. It will attach directly to the antiligand substrate in an amount corresponding to the amount. Also, the ligand of the sample treated with labeled ligands and antiligands substituted by an amount proportional to the amount of It is also possible to include the substrate in a probe formed from the treated substrate.

The present invention has yet another excellent effect. For example, carrier particles can be used as opaque patches When assembled as a complex of substrate, antiligand, ligand, and probe, In particular, the amount of water from the analyte-tagged anti-analyte product of interest is It provides an extremely effective baseline against which even light from small meals can be detected.

A further effect of this detection method is that it can be It does not require precise optical lenses or optical materials to perform detection through There are many things. In fact, you can focus at any suitable angle on the wall of the vessel selected for detection. It is known that it works effectively with light that is focused on points. In many situations, N-ko and Hotaru It is most convenient to measure light (epiflu orescene), and desirable. In many cases, containers are used to store separated liquid “foam” for evaluation.9 The inside of a plastic tube, such as a fluorocarbon tube, can be , moving as one of a series of containers.

In the present invention, the term "optically labeled" means labeled with a dye, As a result of the labeling, the labeling results in an immune system that can be detected using suitable light sources and detection means. Used to refer to epidemiological components. Fluorescent dyes are very suitable for labels. p, and other dye raw materials, although techniques for using them are well known in the art. can also be used in some situations. Fluorescence detection technology with living cells Due to their superior coexistence ability and sensitivity, many embodiments of the present invention They have become the preferred technology. Other tags, e.g. radioisotope tags If a probe is used to form a probe, an appropriate method of detecting radiation may be used. Wear. Labeling can be carried out in any suitable way - for example fluorescent substances 22 sets of glass or plastic beads (i.e., the final (which can be attracted to probe beads), then place the beads inside the There is a method of coating the probe with powder to form a complete probe.

Also, a fluorescent tag is attached to a secondary bead to form a probe bead, and then, Probes can also be formed by treatment with antiligands.

Additionally, primary glass beads can be used in combination with magnetic beads as a substrate material. You can also do that. Measurements of different parameters can be performed on each type of bead (e.g. This can be done with different antiligands on the beads.

One of the claimed advantages of the present invention is that it can be performed on biochemicals to be detected that are measured in the field. That is, it is carried out in the medium in which the substance to be detected is produced. vinegar That is, the volume within the container (i.e., chamber) containing the organism to be detected is substantially There is no physical damage at all. This means that the volume being analyzed is very small; That is, it becomes very important in the case of 0.25 to 10 microliters.

As is clear from FIG. 7, the detection and analysis technology of the present invention allows secretory cells to grow and Coproteins or glycolipids or carboidronates are secreted by cells. can be carried out in a small, closed chamber containing trace amounts of biological compounds Wear. In a typical situation, the vessel or reactor is 18 gauge, i.e., with an internal diameter of 0. ．． FEP (Fluorinated Ethylene) be confined within a polyfluorocarbon tube made of Ru. Such tubes have an inner diameter of about 0.03 to 0.04 inches and a wall thickness of about 0. ．． 01 to 0.02 inches is Zeus Industrial Pr0du By CtS+Inc, (Address: Rantan, New Jersey) It is sold under the product name rZeus J. The tube is sterilizable and non-toxic. Not wettable by water and permeable to gases.

Early detection of specific monoclonal antibodies is particularly problematic. Appropriate secretion of puberty The presence of cells is also identified by the presence of specific antibodies in the culture medium during sampling. here In this case, a growing and dividing (i.e., in situ) pie capable of secreting antibodies In the presence of hybridomas, homologous detection of antibodies allows the detection of specific monoclonal antibodies. Homogeneous analytical techniques have been described to detect the presence of .

For example, it is extremely important to be able to quickly indicate the presence of cells that secrete monoclonal antibodies. desirable. Such detectability is enhanced by decreasing chamber size, and why secreting cells tend to secrete the same number of antibodies as they would if they were in a larger volume. It's to cool down. Additionally, the chamber can be made smaller and more spherical (or vertically elongated). increases the relative surface area per chamber volume for gas exchange into the chamber. can.

These small dimensions make it difficult for the perfluorocarbon fluid to be dispensed into the tube. This can be achieved by carefully proportioning the volumes of biological culture solutions. In other words, the room By reducing the dimensions and creating an appropriate shape, you can easily concentrate the patch to be evaluated. detected in such chambers (as an indication of the presence of secretory cells). The concentration of monoclonal antibodies used can be increased to enhance detectability. Book The invention has already been selected for its ability to express monoclonal antibodies The antibodies being produced by secretory hybridoma cells are described in detail below. However, the present invention provides for the production of hybridoma cells for use in monoclonal products. It can also be used to detect the presence of specific antibodies formed in the method of choice. Ru.

The method specifically described herein is performed in a chamber of the type described in the illustrated embodiment. to ensure that there are fewer than 10 secreting cells per 10 microliters of culture medium. It has a very good ability to detect.

Antibodies can be detected even at concentrations below 40 nanograms per milliliter.

As will be clear to those skilled in the art, particle patching or other types of Measuring radiation is not the only way to utilize the invention. There tags, fluorescent or other labels that remain in the liquid phase after the particles are extracted from the The amount of water is also an inverse measure of the amount of material attracted or retained in the "patch" area. Can be used. In this method, the radiation signature of the tag is substantially different from the background. You need to be different too.

Alternatively, the first analyte to be detected may be added to the separated substrate by magnetic or gravitational means. The different analytes to be detected can be separated using different methods. be done. In other words, some substances to be detected are separated using magnetic technology, while others are separated using gravity. can be released. For example, the substance to be analyzed can be determined by optical measurement. remain dispersed in bound form within the medium to be analyzed; Physical separation can also be achieved by technology. This last technique tags fluorescent background (biologically generated) fluorescence is very low. This is particularly useful in cases where

All of these methods have been described by experts in biological treatment methods known in the art. This can be easily implemented by reading the disclosure details.

For the sake of clarity, a single antiligand, i.e., a specific What aids in the detection of fluorescent antibody ligands and the light emitted from a single fluorescent source? The present invention will be described with reference to a case where the following is used.

However, as a matter of course, the present invention allows two or more antibodies to be combined with different antiligands. It can also be used in a way that allows both to be detected at the same time. each The antiligand is another label (e.g., a different fluorescent tag) that is optically distinguishable from each other by means (i.e., different labels emit or reflect different The probe may have a label (identifiable by wavelength).

In addition, a substantial effect of the method of the present invention is that particles to be analyzed are removed from the chamber before analysis. It may also be possible to implement For example, after removal, a patch can be used without a cleaning step. It is also possible to optically evaluate the problem.

Concentration enhancement A particularly valuable variant of the method of the invention is the early detection of secretory cells of interest. can be carried out very easily, in which the pore network of the solid phase matrix is By combining all secretory products of interest with the cells themselves, the "shrinkage effect" is used. The matrix is composed of opaque antiligand-retaining particles or Can be formed from beads. The main purpose of such a matrix is to limit motility together with the analyte in the immediate vicinity of cells labeled with multiple probes. of analytes that are concentrated nearby (e.g., analytes formed by cells). The goal is to maximize concentration. This allows specific secretory cells or such cells to The presence of a person can be identified very quickly.

In reality, the matrix has a void, i.e. a liquid inside the matrix. There is a space filled with. Making the space smaller allows the secretion immediately surrounding the cell to This is preferable in the sense that it is easy to increase the concentration of the produced product. of the patch The porosity must be large enough to allow the ligand and probe to diffuse through it. Must be. However, the most favorable level of porosity depends on cell size and analyte. Related to factors such as size, quality, and nature of the proof.

It is emphasized that the entire cell does not have to be within the matrix. for example , the cell diameter may be larger than the matrix thickness. In such cases, Preferably, the majority of the cell volume is contained within the mass.

Before reacting with the analyte, paint or other common methods The cells themselves can also contain substrates (e.g., antiligand-retaining beads and probes). If it is found that the patch can be placed within a pre-formed patch substrate (such as a coated patch that can be Ru. When cells secrete the desired antibody, the antibody immediately coats the antiligand. The fluorescent probe material is attached to the substrate, followed by the application of fluorescent probe material, which allows The presence of cells at is most easily detectable. The probe-retaining antibody is You will be relatively focused on your surroundings. Often they form a "halo" effect The reason for this is that the analyte secretion product (actually the ligand-analyte, typically (in some cases monoclonal), but the process of dilution in larger culture samples This is because it is not slowed down or obscured, but attached to the proof. sand That is, the solid substrate matrix limits the amount of liquid in the area being examined and also Analytes (or simple cells) and their production in liquids (usually culture media) in It seems to promote higher concentration with substances. In such a method, The motility of the vesicles and their secreted products is reduced and at the same time their detectability is increased. this Using the method, single cell production (e.g. the desired monoclonal secreted product) can also be detected. Alternatively, the secretory cells may be part of a patch. such a place If so, the proof can be removed while still contained in the patch, or added after positioning the patch. may be part of the added liquid composition.

An excellent way to achieve this high-density effect is, for example, with magnetic beads. It was discovered that there are different ways to bring cell populations into close contact with antiligand-treated beads. It is. Cells and anti-analytes to be monitored for analyte products The substrate mixture with the retention beads is placed on the reactor surface, i.e. in a 96 well On the inner surface of the tubular chamber, before supplying the residual culture to the bottom chamber, such as a crotitre plate. Can be coated. Alternatively, anti-analyte-retaining beads can be It can also be coated on the reactor surface before adding the cells to the top of the reactor.

Also, of course, on the surface of the wells of a so-called 96-well plate and, for example, on clear optical surfaces. Cells can also be stacked directly above or below the top porous patch of beads. .

Another method for forming and utilizing cell/bead patches is to Place it on the film and immobilize it with a gel coating if necessary; After the area with the body or secreting cells has been identified, part of the cell intake process is carried out. There is a method of cutting out that range and removing it. The seat is especially convenient The device specifically allows for the opening and recovery of areas containing the desired secretory cells. can be done.

For all of these treatments, the analyte-retaining patch is located in close proximity to the transparent surface. where it can be monitored by light (most preferably shrimp reflected light or shrimp fluorescence). You will be able to do it.

Opaque beads are not coated with antiligand in addition to being used as substrates. Sometimes it also serves as a background shielding agent.

In this example, instead, a microtiter plate or a transparent film or membrane is used. On the substrate, the antiligand is coated.

Depending on the optical density of the specific test system, it may become opaque after reacting with the probe. Pull the patch to the backside of a cell layer or other non-opaque substrate patch, thereby It may be desirable to shield the detection system from background radiation. So Opaque patches such as are particularly useful in shrimp reflective or shrimp fluorescent systems. , is completely useless in light transmission systems. In such reactions, opaque patch-shaped It is not necessary to coat the particles with biochemicals.

Opaque shielding, which is commonly performed with magnetic beads, is difficult to achieve even after the reaction of the analyte. It can be effectively carried out using "pre-positioned patches". In such a situation , the original culture medium and the basic substances used in the invention are placed in a measuring container. secondary bee groups (commonly used for pre-positioned or “painted” bottom patches) A bead array (common magnetic beads) is added to the culture medium to create a patch. cover.

This second population of opaque beads can then be used (e.g., magnetically or without the need for magnetic forces). (if appropriate, gravity) on the back of the painted patch or immobilized substrate. of any optical parameter used in the fluorescence or phosphorescence or detection process. During the measurement of light, it is shielded from background light radiation in the medium.

In this regard, the above-mentioned patient tumor cell phagocytosis method uses a magnetic vibrator after background reaction. These are typical situations that can be covered by the virus. Of course, all reactants are in place When in position, an opaque shielding patch can be drawn after them before reacting. this In this case, the diffusion and porosity in this shielding patch will also be reduced throughout the patch and most importantly Labeling is allowed to occur at the photostimulated surface.

Particularly effective is the ability to form a layer containing antigen (anti-analyte) on the substrate. i.e., the substrate of the 96-well plate covers the anti-analyte layer with opaque beads. , then only the cell retention medium containing the antiligand-tagged plume is added to the bead. added to the water. All analytes secreted from the cells are Find a path to Anti-Anara and Ito through the elements. The proofing layer is It can attach to the analyte either before, during or after the passage. opaque When using bright beads only for shielding, block the surface with BSA etc. and It is most effective to avoid undesirable attachment of biochemicals to

The present invention can be used to analyze intercellular functions, such as for use in cell surface analysis or analysis. Both are possible. That is, the internal Cell markers may also be used. They cannot be detected until they enter the cellular environment. Tag substances without tags can be used. The resulting marker proof is The cellular functions of the cells are shown qualitatively and quantitatively. With such markers available There are enzyme markers, calcium markers, and 1)) (shape?-force-).

Many such markers are available from Calbiochem Immunochemi. cals, Behring Diagnostics, and Divis available from Hoechst Corporation.

Similarly, cells interact with DNA, such as DA sold by ence Inc, of Washjngton+PA DN such as PI or Calbiochem Immunichemical) A? - It is also possible to label cells intercellularly with a dye that serves as a marker.

In one embodiment of the invention, microtiter wells are coated with a series of substances. So The material contains the culture medium in the test for the analyte. under culture medium The lower wall of the cell, which can be based on beads or some other substrate, is then anchored. A thiligand-retaining layer is applied. The middle between these two layers is optical and very thin. It can be layered or it can be an immobile gel such as Aga gel.

Of course, there is no need to dust the surface with a microtiter well. The important points of the present invention are: The sensitivity of the invention is due to the use of shrimp fluorescence.

Some of the embodiments of the invention relate to cells in pre-placed patches. In this case, a population of cells can act as a substrate, e.g. an analyte substrate. Wear. In such applications, place the opaque screen behind the patch, e.g. It can be formed on the opposite side. In such applications, tumor cells can be removed from the punch. A panel of monoclonal antibodies can be provided as a shield to bind to the surface. If desired, use batches of tumor cells as targets for detection through Place them as a substrate next to the surface of the transparent container you are going to perform. In fact, tumor cells A number of samples of cells were placed in different chambers, i.e., in a 96-well microtiter plate. It will be added to the file.

Each well was then injected with a different cell surface antigen.

Adding different monoclonal antibodies known to each correspond to pe Ru. When a monoclonal antibody binds to a patient's tumor cells, it binds to a group (e.g. fluorescent It acts on light, anti-antibodies) and merges it with the cell surface. This cell combined fluorescence is Next, it is preferably measured by shrimp fluorescence. As explained later, the optical range Limit the depth of the points or use shielding particles to arch them to the back side of the cell. In this application, the invention is limited to cell matrix patches by forming an opaque screen. describes a preferred embodiment of the invention, and suggests various variations and modifications thereof. However, the present invention is not limited thereto, and within the scope of the present invention Other changes and transformations may also be made. What is suggested here is It has been selected and included for the sake of clarity, and it is also possible that a person skilled in the art would understand the present invention and its effects. Be able to fully understand the process, modify it and implement it in various forms, and This is so that it can be adapted to the situation.

Brief description of the drawing Figure 1 is isolated in a chamber within a plastic tube, shown most clearly in Figure 8. FIG.

Figure 2 shows the same culture as shown in Figure 1 after the antibodies have been expressed by the M5 cells. It is a diagram.

Figure 3 shows the same image after both the antibody and fluorescent probe have been substantially bound to the magnetic beads. It is a figure showing a culture solution.

Figures 4, 5 and 6 show magnetic material drawn into the opaque patch on the side of the chamber. FIG.

FIG. 7 is a schematic diagram of the light collection and measurement system.

Figure 8 shows a typical tube handling device for handling a range of biochemical complexes. This is a schematic diagram.

FIG. 9 schematically shows the preferred shape of the chamber in which the method of the invention is carried out in the most preferred manner. It is a diagram.

In the following examples related to Figures 1 to 6, a pibridomoma is In situ allogeneic analysis to detect the presence of secreted antibodies (by cells) is demonstrated. . The tubular structure of the reactor chamber is described in U.S. Pat. No. 3,491.141 (inventor: Sym It is similar to that adopted by the et al. Sterilize the tube properly and overfill. Use an orocarbon carrier liquid.

Example 1 This analysis demonstrates the detection of antibodies with dimensions greater than 100,000 Daltons. be.

As shown in Figure 1, the polystyrene magnetic beads 2o have a nominal diameter of 1.75 micrometers. It is supplied by Seragen and is suitable for use in water, absorption, boat, etc. Anti-mouse (CAM)-coated engineered GG antibody (sold by Zymea) (heavy and light chain formats) are used. Using bovine serum albumin (BSA) Then, the other reaction sites on the magnetic beads were closed. Beads 20 are for probes. The probe is bound to the bead and ultimately becomes opaque. drawn into the batch. The beads are separated into a tube-coupled reactor, i.e., chamber 22. The chamber 22 contains a tissue culture medium 24. There is a so-called tap in room 22. The chemical or probe 26 is also dispersed 9, and the probe 26 is from Zymed. A FITC-tagged boat anti-mouse cloned antibody is available.

In addition, pre-selected hybridoma cells 28 (engineering gc, 2a) are generated 55 ．． 2 bipridoma cells) are also dispersed in chamber 22. Before use, cells should be diluted approximately 4 times. Wash with Hank's salt solution to remove all free antibodies. In another room there is a negative control Non-secreting 653 mouse myeloma cells were used as control cells or as positive antibody control cells. Contains either gG2a mouse myeloma protein (a product of ICN). ing.

Figure 2 shows a new situation in which, after a certain period of time, the antibody 30 is secreted by at least some cells 28 (i.e. secretory cells) Ru.

Magnetically coated with boat anti-mouse antibody (also called M Qi Beads 20 "Prove" FITC Tag Phrase Goat Anti-Mouse Compound Claw Similar to sexual antibody 26 (also called GAM), newly secreted antibody 30 (also called Ab) call). The resulting composite is shown in Figure 3 using the nomenclature above. is shown as chemical compound 32 of the following formula:

MB/G AM-Ab-FITC/GAM probe antibody 26 was Because the antibodies are present, they simply merge with the beads.

As shown in FIGS. 4 to 6, the cornerstone 40 has dimensions of approximately I x 1.4 x 7 centimeters. Chimedor's 7 kilogauss magnet, which is used to transport magnetic particle-retaining materials into a chamber. 22 to a small area around it, and then create a patch as seen in Figure 6. 42, forming a generally opaque mass of transparent beads.

The amount of fluorescence emitted in response to appropriately matched stimulus light as known in the art is: Not necessarily secreted by cell 28 and MB/G AM-A b-FITC/G Anti-resistance attached to both edges of MB/GAM and FITC/GAM to form AM substance It is not directly related to body mass. Of course, it's still attached to the antibody. If there is some magnetic material, it is also placed in the magnetic patch 42, but it is not a fluorescent material. It does not contribute to light emission.

In typical operation, the fluorescence used to measure the amount of labeled product is Creases are generated and measured using a microscope. The microscope includes a fluorescence source means for quantitatively measuring fluorescence. , i.e. it is equipped with a multiplier phototube (PMT) and suitable readout means. General Microscopes usually include multiple boats containing receptors such as PMTs, as described below. is the provision. Note that this example is for the case where FITC is used. other than that For most light sources, using different emission labels and different best excitation wavenumbers is most effective. Can be used for

Other optical systems are also useful in detecting the presence of probes in the path. be. For example, using an argon laser 46 (with a wavelength of 488 nanometers) ) generates a light beam 66 that passes through a filter 68 to remove the bore light and all That is, the incoherent light normally emitted by such lasers is eliminated. That's it The resulting beam undergoes beam expansion and collimation. (mating) The beam passes through an optical lens combination 70 and becomes an arbitrary beam diameter. So For example, the diameter of the patch 72 is approximately 10 microns and 6p, in a typical example. , the average diameter of which is collected by magnet 84 is about 100 microns. on the patch The specific dimensions of the beam prevent excessive irreversible "bleaching" of the fluorescent probe (fluorescence loss of radiation ability) so that it is strong enough to rotate watts/c+++2) selected.

The beam is reflected from the dichroic beam splitter 76 at a 45 degree angle and then the beam It passes through an objective lens 75 which focuses 74 . The beam splitter 76 is It reflects more than 90% of the argon laser beam 66 and transmits less than 10%. is selected. Additionally, the beam splitter is responsive to the fluorescent stimulation beam 74. Fluorescence spectrum emitted from the patch (focused at about 518 nanometers) It allows more than 90 wavelengths to pass through and reflects less than 10% of those wavelengths. released from the patch This emitted fluorescence 78 passes through an objective lens 75 that makes the light parallel, and then the beam It passes through a splitter 76. Beam 78 is a heavily reflected beam of stimulation beam 74. For this purpose, a barrier filter is applied before passing through another lens 81. -80. The lens 81 generally focuses the collected light. is aligned with a detector 82 which is a silicon photodiode. Photodiode output The power is proportional to the amount of light in beam 78 and is amplified into an output signal.

Having read the above description, one skilled in the art will be able to identify the appropriate hardware, i.e. Zu! Filters, mirrors, amplifier circuits, and signal output devices can be easily selected.

Of particular note are the non-reactive fluorescent probes and the tissue culture medium itself. The overall background fluorescence generated by patch 42 is due to the opaque nature of the magnetic patch. They are shielded so that they have no substantial effect on the light reflected from them. especially the patch The means for introducing light into the optical system can include a number of processes known in the optical arts. , optical fibers are used to transport light to and from the patch for analysis. It can also include one means.

FIG. 8 is a schematic diagram of a typical continuous processing process utilizing commonly known concepts, including According to this, a chamber 22 with a volume of about 10 microliters, which is the object of analysis, is filled with air bubbles 50. While separated, they are moved within a polytetrafluoroethylene tube 52. . The liquid material is a perfluorocarbon liquid compound, manufactured by 3M Company as Fluortnert. It is sold under the trade name FC-77, and the substance facilitates separation. At the same time, the reactor is sufficiently lubricated along the inner wall of the reaction tube 52. It forms a means for continuous and easy movement. Tube 52 has sufficient It forms a means through which nutrient gases and CO2 can pass.

A gas or air bubble between the chambers surrounds the double edges of the chamber containing the gas bubble and the biological culture medium. Provides another surface for gas to pass through the luorinert carrier phase 60 be able to. Clinical work will be conducted according to the following guidelines.

Add the coated and occluded magnetic beads to a 96-well tissue culture site. that place may contain either secretory or non-secretory cells, or, in the case of experimental control, G2a or nutrient medium (or "tissue culture" medium).

All chambers contain treated magnetic beads approximately 1.12 Contains a typical concentration of 10 beads. The concentration of beads can be further reduced. . The probe has a concentration of 375 nanograms of FITC/GAM per milliliter. has been added. 4 to 250 hybridoma or non-secreting myeloma cells are initially placed in a suitable reaction chamber. IGG2a mouse myeloma protein supplied by ICN Lotin can also be used as a positive control in combination with magnetic beads and fluorescent probes. .

It was placed in a separate reactor at a concentration of 37.5 nanograms/ml.

The interior of the tube was lightly coated with carrier liquid.

The fluid was poured with Fluorinent FC-77, and the tube was poured stably. Wetting, gas permeability, and appropriate permeability to allow optical analysis. It is a compound that has Transfer the reaction mixture from separate wells to the hema in Teflon tubes. Inhale using a ni-fold and syringe pump system, then insert the tube A reaction chamber was formed inside. Sufficient FC-77 is also fed into the tube by the bomb action. A barrier layer was formed in the chamber of the biological culture medium, which was the reaction mixture. The relevant technology Air was introduced into the tube to separate adjacent chambers, as is known in the art. Next The chamber inside the tube was placed in an incubator at 37°C and 5% CO2 in a 95% air environment for 24 hours. I put it there. A magnetic bead is then drawn into the opaque patch, causing the patch to become fluorescent. Measured.

All reactors from wells containing either secreting cells or engineered gG2a are fluorescently It was positive regarding luminescence. A chamber containing only non-secreting cells or medium is I lost (negative) against fluorescence.

The above example uses substrate-independent cells. Also, matrix-dependent cells such as liver cells and There are also fibroblasts and fibroblasts.

They are known in the art and sold by New Brunswick. must first be attached to a suitable microcarrier.

Example 2 Each step of Example 1 was repeated using particles with relatively high transparency instead of magnetic particles, i.e. , was repeated using glass beads. The glass peas fall into the ground due to gravity. Separated. If the stem is thin enough to allow light to pass through, the collected light will Care must be taken to ensure that only those from Chi. Excitation beam and emission beam This effect can be achieved by tilting the beam to some extent.

Example 3 Two or more properties of a single ligand can be detected using multiple different probes It is also important that

That is, for example, if a certain analyte tends to be specific for one antiligand, There are cases where you want to prove that other things do not have special effects.

The labels may then still be of the same general type, e.g. two different fluorescent tags. stomach. For example, one group can be used to create antiligand characteristics for a ligand. Detect the fluorescence spectrum resulting from the efficacy and use another probe to probe the ligand. It is possible to measure the lath and the same reference specimen.

In other words, hybridization to human hemoglobin (α chain, not β chain) Doma-secreted antibodies can be selected using the following procedure. GAMr-1 with a magnetic beam used as a coating on a wafer substrate. Probe 1 is fluorescent tag reference 1 (i.e. FI TC) with the hemoglobin α chain of the person labeled. Probe 2 is a fluorescent lamp. Human hemoglobin beta chain labeled with 2-glucose 2 (e.g. Texas Red) It is. And secreted by bilidoma cells that are specific to human hemoglobin alpha chain. IgG, antibodies must be identified in the chamber where the patch is tagged 411 In Tagsha 2, there is no labeling at all, ( A cell population that secretes antibodies with the specific efficacy of Proube-san 1 (not Proube-san 2) exists. In cells where the antibody is specific for Probe San 2, the opposite is true. applies. In the third case, all rooms where the double-edged label is detected by the patch In other words, a mixture of cells that are specific to each of the two probes, or a mixture of cells that are specific to each of the two probes. This refers to antibodies that are highly effective against antibodies. To distinguish between these possibilities, the third In some cases, cell clones of the chamber are formed in the same system. That is, the present invention According to υ, cells spread between different chambers, so that only one cell per chamber comes to exist. Each chamber was retested using each of the two groups. and all chambers that are specific to only one probe are measured.

FIG. 9 is a schematic diagram showing a generally spherical chamber 91 within a plastic tube 93. The tube 93 is separated by an air bubble 92, which facilitates separation of the chamber and air bubble. 93 and facilitate the movement of air bubbles, particularly the movement of chambers along the tube 93. Contains a fluorocarbon liquid 94.

A typical procedure in which concentration effect 1 can be observed is as follows: do. Using sterile techniques, sterilize the following reagent solutions in 3.5-4. Add oul.

Media: RPMI 1640 with 20% FC3 or 20% FCS Optimurn’.

Boat anti-mouse coated latex calm beads 5: diameter 0.7mm, density 1.9 5 g/ml, or diameter 1.7 mm, density 1.07 g/ml, concentration 0. 0010 sw/V~o, 003%w/v 0 Goat Anti Mouse FITC 6: Density 0.375mg/rnl~1. s m g / m 1 a time), RPMI 1640+ at a hybridoma cell concentration of 25 cells/well Mouse bipre consisting of 20% FC3 or Optimum + 20% FC8 Add 3.5 ml of Doma cell solution.

Approximately 3 to 24 hours after adding the cells, a halo of fluorescence 7 was observed in the GLM. In the BM they can be seen surrounding the mouse bilidoma cells, which are themselves surrounded. I can do it. GLM Cells that are not in the pellet of MB are located on the beads closest to them. act on the edges of the cell to make it luminescent Hen,a, within 3-24 hours after cell addition. Fluorescence emission occurs as a "hot spot" in and around the GLM MB pellet. Can be seen. Additionally, 24-48 hours after cell addition, the “hot spots” become soluble. This results in an overall pellet fluorescence emission.

by mouse hybridoma cells in or near the IGLMMB pellet. Binding of secreted mouse antibodies and binding of free GLM FITC to mouse antibodies Fluorescence emission caused by

2L45-50 oil - Union Carbide silicone oil "Terasa" ki board-Robbins 5cjentjfic 'JJ' Optimum- Manufactured by Gib 5Latexa Beads - 5eradyn 6 Boat and Mouse FITC-B Fluorescence emission that can be observed with the iomedia 701Ympus IMT2 fluorescence microscope In addition, the following claims cover the general and specific &! of the invention described herein. all the signs , as well as all explanations of the scope of the invention contained therein. It is.

Engraving (no changes to the content) ■ pole Procedure amendment writing method) Heisei Year Month Day 3.2. −(3

Claims (1)

[Claims] 1. Formed in a substrate means in a liquid compound placed in a chamber and made detectable a labeled first antiligand-ligand product; It is a seed method, (a) the detectably labeled first antiligand-ligand product; carrier particles coated with a second antiligand in the same manner as the above substrate means for established; (b) the above retained by said carrier particles from said liquid in said chamber in batches; at least a portion of a detectably labeled antiligand-ligand product; gather; (c) a step for measuring the amount of detectably labeled product present in said batch; A method characterized by comprising a step. 2. Light from at least a portion of said compound in said room from said batch under evaluation. The above carrier is used to shield the above room from scientific background interference. 2. The method of claim 1, wherein said batch is measured during said batch. 3. Optical background drying from the above compound in the room from the above range during detection. Claim 2, wherein the evaluation is performed using an opaque carrier as a means for blocking interference. the method of. 4. Determination of the amount of detectably labeled product is accomplished by the assembly step described above. After substantially releasing the substrate-retaining antiligand-ligand product, the liquid compound is 2. The method of claim 1, wherein the method is performed as an inverse function of the residual proof. 5. The above labeled antiligand/ligand is a labeled nucleic acid supplemented nucleic acid product A method as claimed in the claims. 6. Multiple different detectable labels are used to form a powerful probe. Each probe holds an antiligand that exhibits different properties of the above ligand. The method according to claim 1. 7. The method of claim 1, wherein said particles are magnetic and said batches are opaque. 8. The method according to claim 1, wherein the evaluation is based on an optically detectable label. . 9. 2. The method of claim 1, wherein said patches are opaque. 10. The detectably labeled antiligand-ligand product is Contains Proop beads with fluorescently labeled compounds within the Proup beads The method according to claim 1. 11. Claims 1, 2, 3, 4, 5, wherein the above method is carried out in the presence of living cells. 7. The method according to 6 or 7. 12. The magnetic particles are applied to the opaque batch by the magnetic field before the evaluation step. 10. The method according to claim 9, wherein: 13. 2. The method of claim 1, wherein said gathering step is performed by gravity or centrifugal force. 14. Claims 2, 3, 4, 5, wherein the gathering is performed along a transparent area of the wall of the chamber. Or the method described in 6. 15. at least one optically labeled antiligand/ligand product as described above; Claim 1, 2, 3, 4, 5 or 9, wherein the labeling is carried out with a fluorescent label of How to put it on. 16. As an additional left step: (a) the label to be detected from the batch; (b) redispersing the substrate-retaining antiligand/ligand material; Later, a step of reassembling and optically reevaluating the substance for another optical evaluation. 10. The method according to claim 1, 2, 3, 4, 5 or 9, comprising a step. 17. The above ligand-antiligand complex to be detected is a monoclonal antibody. The method according to any one of claims 1 to 10, comprising: 18. The above ligand-antiligand complex to be detected is a monoclonal antibody. and the density of the cells in the chamber that secreted the antibody is above 10 microliters. less than 10 cells per culture medium, and the density of the monoclonal antibody is 13. The method of claim 12, wherein the amount is less than 40 nanograms per . 19. the first antiligand retained on the substrate and the second antiligand 2. The method of claim 1, wherein both antibodies are monoclonal antibodies. 21. the first antiligand retained on the substrate and the second antiligand 2. The method according to claim 1, wherein both antibodies are polyclonal antibodies. 22. the first antiligand is polyclonal or monoclonal, and the first antiligand is polyclonal or monoclonal; 2 antiligands are polyclonal if the first antiligand is monoclonal. and if the first antiligand is monoclonal, the claim is monoclonal. The method described in 1. 23. The dimensions of the chamber are approximately 1 microliter or less, and the horizontal dimension is substantially 2. The method of claim 1, wherein the method is non-elongated. 24. Amount of known ligand using probe with known ligand and label Competitive evaluation of samples to be tested for target or qualitative quantity: (1) The ligand is initially present in the sample under test through the known ligand compound. will attach to the antiligand-retaining substrate only in an amount that depends on the magnitude of the competing amount of the ligand. and (2) Remove the above probe attached to the antiligand-retaining substrate in batches. (3) reversal of the presence and amount of competing ligand initially present in the sample under test; The method is characterized in that the probe attached to the antiligand substrate is detected as a measurement. analysis method. 25. Analysis method using labeled probes containing known ligands and labels (1) the probe is labeled with a substrate treated with an antiligand; (2) the sample to be tested for the presence of the ligand; Add to the above bupup; (3) Only an amount of the probe-retaining substrate is proportional to the amount of ligand available to the sample being tested. replacing the labeled ligand; (4) Collecting the proof-retaining substrate remaining in the batch; (5) a supernatant attached to the antiligand as an inverse measure of the presence or amount of the ligand; An analysis method characterized by detecting a probe. 26. An apparatus for detecting optically labeled chemicals, the apparatus comprising: (a) at least one chamber having a transparent wall portion; (b) a batch within said chamber; means for concentrating said chemical; (c) said transparent wall of said chamber forms a means through which said patch can be observed; (d) The radiation emission or light reflection characteristics of the small area are measured optically; A device characterized in that it is equipped with means for evaluating. 27. The above means of concentrating the above chemical substance as a means of attracting magnetic particles. Once the particles are attracted, they are equipped with a magnet for optical evaluation. forming a means for shielding background radiation from the above means, and a means for forming a substrate for said chemical to be detected; 27. The apparatus of claim 26, further comprising means for holding a light label. 28. the axis of said chamber is perpendicular to the light path of said detection device impinging on said chamber; , said chamber does not extend substantially along said axis thereof, and said method for performing optical evaluation Gravity is used as a means of concentrating the batches in the transparent wall near the steps. 27. The apparatus of claim 26, wherein: 29. The method of claim 1, wherein the ligand detected is greater than 5000 daltons. . 30. The method according to claim 1, wherein the detected ligand exceeds 10,000 daltons. Law. 31. 2. The method of claim 1, wherein said patches are non-opaque. 32. A method for analyzing funalite in a biochemical environment, comprising: (a) immobilizing the antiligand on the substrate; and (b) immobilizing the antiligand on the substrate; Contact the antiligand with the culture medium containing the labeled ligand for the antiligand. Let me; (c) forming a composite substance incorporating the above-mentioned ligand and the above-mentioned antiligand; the substance comprises the antiligand and the ligand bound to the substrate; the law of nature; (d) Evaluating the characteristics of the light reflected or emitted by the above substances using a light evaluation mechanism; A method comprising the step of optically evaluating the properties of the composite material. 33. The substrate is beads, and the method includes some of the small beads, Form into a transparent batch and perform the above optical evaluation step in a small part of the batch. the small part is on the side opposite to the culture medium and the light evaluation step is provided. 33. The method of claim 32, facing towards the device. 34. A method for facilitating homologous detection of secretory cells in a culture medium, the method comprising: (a) forming a sample of the medium in a chamber of less than 10 microliters; (b) culturing said sample for a period of time sufficient for proliferation of said cells; (c) The above-mentioned secretory cells of the above-mentioned cells can be sent to each chamber as an indication of the presence of the above-mentioned secretory cells. A method characterized by detecting a compound. 35. 33. The method of claim 32, wherein the volume of the chamber is less than 1 microliter. 36. 34. The method of claim 33, wherein the volume of the chamber is less than 1 microliter. 37. 35. The method of claim 34, wherein the volume of the chamber is less than 1 microliter. 38. The secreted product is an antibody with a size exceeding 100,000 Daltons. 35. The method of claim 34. 39. The secreted product is an antibody with a size exceeding 100,000 Daltons. 36. The method of claim 35. 40. for detecting anti-analyte-analyte complexes in culture media. A method, wherein the complex comprises at least an antibody conjugated to a detectable label. The cell includes a plume formed of thianalite and a cell, the cell forming the plume. The above method is detected by secretion labeling of the analyte from which it is detected. (a) said pore matrix by positioning a portion of said cell within said pore matrix; By reducing any analyte secretion of the cells and the amount of the culture medium in close proximity to the cells, In both cases, the matrix is formed of immiscible substances and the culture medium and its (b) similarly supported by said label-bearing analyte; , through the use of the pore matrix, the interior and vicinity of the cells and the matrix reduces the motility of any labeled analyte loops within the chain structure and (c) achieving concentration of the labeled analyte plume around the cell; Based on the criterion of whether or not the above cell secretes the above analyte, the above cell Measure the detectable left output of the range of detectable labeled probes in the immediate vicinity of A method characterized by: 41. the cell is adjacent to the surface through which the fluorescence detection is performed; 41. The method according to claim 40. 42. Claim 40, wherein said target area is limited to the surroundings of one said secretory cell. How to put it on. 43. Claim 40, wherein said detection is performed by measuring epifluorescence from the target area. How to put it on. 44. Anti-analyte-analyte complex combined with fluorescent label in culture medium , wherein said probe is initially present in a medium containing said cells. It is left only long enough to be in contact with the analyte. Any binding products of the above probe analytes will form along the transparent surface of the container. and then A few magnetic beads from the above medium are removed from the background fluorescence in the above medium. providing an opaque shield at a position between the above-mentioned combined product and the above-mentioned culture medium; Attract, then, said connection of said probe to said analyte while said shield is in said position; The method is characterized in that epifluorescence of light from the target range is measured as a measurement of the target range. Method. 45. 45. The method of claim 44, wherein the probe and analyte are bound intracellularly. . 46. The method according to claim 45, wherein the probe and the analyte are bound outside the cell. Law. 47. A method for analyzing epifluorescence emitted from a target area, the method comprising: To investigate the target range for the epifluorescence emitted in response to stimulation by In a method comprising a fluorescently tagged antiligand bound by 1. Place the tagged antiligand above on the observation surface, such as the bottom of a transparent plate. forming and covering the 2. drawing an opaque screen of magnetic particles from said light source to the opposite side of said target area; 3. All backgrounds on the side of the opaque screen farthest from the light source. A method characterized in that the target area is shielded from fluorescence. 48. (a) the above-mentioned anti-analyte, or (b) at least a cell from the culture medium. The other is a labeled anchor that is immobilized in a coating in the immediate vicinity of the transparent surface. A method for detecting a thianalyte-analyte composite, the method comprising: 1. The analyte produced by the above cell and the above (a) anti-analyte. React; 2. The analyte anthium by epi-irradiation analysis through the above transparent surface. A method further comprising a step of analyzing the above reaction product for a nalite reaction product. Law. 49. 49. The method of claim 48, wherein said analysis is by epifluorescence. 50. 49. The method of claim 48, wherein said patches are opaque. 51. Before the above analysis step, a 50. The method of claim 49, further comprising the step of pulling the opaque magnetic badge so as to 52. the first antiligand is polyclonal, and the second antiligand 2. The method of claim 1, wherein is monoclonal. 53. The first antiligand is monoclonal, and the second ligand is multiclonal. The method according to claim 1, wherein the method is loan-like.