Improved ligand assay

Description: TECHNICAL FIELD [Detailed description of the invention]

Field of invention The present invention relates to methods and products for detecting the presence of specific compounds in various substances. More specifically, the present invention is faster, easier, and cheaper than techniques conventionally known in the art. It relates to methods and products for detecting biologically important ligands by accurate assay techniques. Background of the invention A great deal of research has been devoted to the development of accurate techniques for determining the presence of substances in question, such as organic substances in food, soil, and body fluids, such as drugs and pollutants. For example, pathological or other conditions in humans and animals are often detected by performing immunoassays on body fluids, such as urine or serum samples. The immunoassay recognizes or binds to a second compound, known as a receptor, which has a specific spatial and / or polar organic tissue. Based on the ability of the first compound, known as the ligand. Typically , immunoassays are used to detect antibodies or antigens in body fluids. An antigen is a foreign substance that, when introduced into a higher animal, reacts with the antigen to form an antibody that initiates protection against infection or disease. Antigens or antibodies can be confirmed or determined by adding the corresponding antibody or antigen to a sample of body fluid. The presence or absence of an antibody or antigen in this sample is usually established by detecting the occurrence or non-occurrence of a reaction between a particular ligand / receptor pair, which usually manifests itself by insolubility or aggregation. Will be done. Since the detection of most ligand / receptor pairs is difficult, it is often necessary to use some inert carrier moiety to facilitate detection. For example, in most latex agglutination techniques, the receptor is covalently bonded to individual latex particles having a diameter of about 0.01 to about 100 micrometers. These particles are cross-linked by complementary ligands or otherwise aggregated. Then, agglomeration of such particles into relatively large agglomerates is observed. Different types of latex agglutination techniques currently known in the art can be divided into three basic classes based on the specific methods used to detect ligand / particle agglutinations. For example, U.S. Pat. No. 4,738,932 to Yabusaki describes a centrifugation technique that involves rotating agglutinating slides on a serological rotator and then using a magnifying device to inspect the wells of the slides for agglutination. It is disclosed. The second class consists of techniques for detecting agglomerates by counting particles. For example, Masson et al., Methods in Enzymology, 1981, 74, 106-139, used non-aggregating particles using complex equipment using forward light scattering. An immunoassay technique for counting is disclosed. Thus, both centrifugal and particle counting techniques have the drawbacks of being complex, time consuming procedures and expensive, highly specialized equipment. The third class technology is a technology for visually detecting agglomerates. However, most visual agglutination tests typically require an undesiredly long time to form, as the average human eye can only detect small particles up to about 40 micrometers in diameter. A relatively large agglomerate must be produced. U.S. Pat. No. 4,459,361 to Gefter discloses a slightly improved type of visual technique that involves the visual detection of non-aggregated particles rather than aggregated particles. In Gefter's technique, both the ligand and the receptor are separately immobilized on latex particles and then mixed with a sample of body fluid suspected of containing the ligand. According to Geftr, the ligand in the sample competes with the particle with the ligand and the particle with the receptor for the receptor site. The ligand-bearing particles can aggregate to the extent that the ligand contained in the sample binds to the receptor-bearing particles. When such a mixture is exposed to a filter with a controlled pore size, the amount of non-aggregating ligand-bearing particles that pass through the filter is substantially increased. What is then detected is the presence of these non-aggregated particles. According to Gefter, the amount of non-aggregating ligand-bearing particles that pass through the filter is proportional to the amount of ligand in the sample. Moreover, as Gefter states, the number of non-aggregated particles is sufficient to be visible to the naked eye, and this visibility is increased by the choice of particle size color, optical density or fluorescence. can do. However, the technology disclosed by Gefter has a number of significant drawbacks. For example, this technique requires the preparation of both ligand-bearing particles and receptor-bearing particles, thus adding considerable time and expense. Gefter claims that particles with non-aggregating ligands can be detected with the naked eye, but the examples provided do not detect the visibility of such particles, but rather complex spectrophotometricity. It is detected by the measuring device. Therefore, there has long been a demand for relatively simple and inexpensive techniques for accurately detecting biologically important compounds present in body fluids and other sample fluids. An object of the present invention Therefore, one object of the invention is to provide methods and components for detecting the presence of compounds in various substances. Another object of the present invention is to provide a method and a product for accurately detecting a biologically important ligand. It should be noted that another object of the present invention is to provide a method and a product for detecting a biologically important ligand, which is faster, easier, and cheaper than the techniques known in this field. .. Another object of the present invention is to provide improved methods and products for performing an immunoassay of latex agglutination. Abstract of the invention These and other objectives are achieved by the present invention providing methods and devices for detecting the presence of a ligand in a sample suspected of containing the ligand. The method and apparatus rely on color visualization to indicate the presence and absence of ligand in the sample. This color visualization does not require complex instrumentation or equipment. All color changes are easily detected by the naked human eye. A preferred method of the present invention consists of forming a test mixture by contacting the sample with colored particles having a receptor specific for the ligand on the surface. Thus, the particles have the ability to form aggregates of the ligand and the particles (or labeled ligand / particles) when in contact with the ligand. The test mixture is then passed through a filter to remove agglomerates from the filtrate, where the filter has openings larger than the particles but generally smaller than the agglomerates. The color of the filtrate is then analyzed. The presence of the ligand in the sample is established when the color of the filtrate is substantially different from the color of the particles having the receptor. The present invention also provides assay plates and reaction cells suitable for practicing the disclosed methods. A preferred assay plate consists of a superstructure having a filter well and an observation well. The assay plate is further adjacent to the superstructure and extends across the filter wells, the core means adjacent to the filter means and extends the length of the filter wells and observation wells; and the suction means ("core means"). Consists of bottom means adjacent to. In a preferred embodiment, the top member, filter means, core means and bottom means are held in place with a suitable adhesive. The reaction cell according to the present invention consists of particles having a receptor. Preferably, the reaction cell is a pipette, and the particles are colored and contained in a destructible container within the pipette. In certain other embodiments, the reaction cell further comprises a killing solution that inactivates all biologically active substances used in performing the assay. These methods can generally be performed more rapidly than other visually detected immunoassays. This is because the relatively small aggregates that indicate the presence of the ligand in the present invention form more rapidly than the larger particles required by the prior art. Also, these methods and devices are as sensitive as prior art immunoassay techniques, but do not require complex, time-consuming operating steps, and do not require expensive, complex equipment. Thus, the methods and devices of the present invention are conveniently used by those who have little or no technical training. A brief description of the drawing FIG. 1 is an upper plan view of the assay plate according to the present invention. FIG. 2 is an exploded cross-sectional view of the assay plate according to the present invention. FIG. 3 is an exploded cross-sectional view of the assay plate according to the present invention having a barrier between the core means and the bottom means. Detailed description of the invention The present invention provides a sensitive yet simple method for determining a wide variety of ligands at relatively low concentrations that may be present in a variety of sampled materials. The present invention can be applied to detect virtually any ligand contained in a sample of body fluids derived from mammals, especially humans, such as urine, serum, and plasma. As will be understood, the term "ligand" means all components in body fluids, cell extracts and tissue extracts that have or can form a partner for an immunological response. To do. Both antigens and antibodies, as well as amides, amino acids, peptides, proteins, lipoproteins, glycoproteins, sterols, steroids, lipids, nucleic acids, enzymes, hormones, vitamins, polysaccharides, and alkaloids are ligands according to the invention. is there. A representative example of an antigen is the "substance" described in US Pat. No. 4,256,834 by Zuk et al., The disclosure of which is added here by citation. The antigens in question include: tetrahydrocannabinol and its metabolites, cocaine and its metabolites, morphin and other achen agents, amphetamines, phosphatases, barbiturates, steroids, rheumatic factors, histoplasma, human chorionic villi. Gonadotropin, luteinizing hormone, vesicle stimulating hormone, C-reactive protein, and acid phosphatase. Of particular interest include: HIV-virus, cytomegalovirus, HIV-2 virus, hepatitis B virus, hepatitis C virus, herpes 1 and herpes 2 viruses, HTLV-1 virus, chlamydiavirus: , Borrelia burgdirferi, Treponema pallidum, Neisseria gonorrhaeae, stapylococcus, and streptococci of groups A and B. Preferred ligands according to the invention are those having more than one active site. As those skilled in the art will understand, the active site of the ligand is the portion that binds to the ligand receptor. A preferred type of ligand with more than one active site is an antibody against the antigen. Particularly preferred ligands are antibodies against antigenic proteins derived from HIV-1 virus, cytomegalovirus and Borrelia burgdirferi. According to one aspect of the invention, samples suspected of containing the ligand in question are brought into contact with particles having a ligand-specific receptor on their surface. Thus, particles with receptors have the ability to form ligand / particle agglomerates when in contact with the ligand. The amount of particles with receptors that come into contact with the sample is preferably selected so that essentially all particles with such receptors form aggregates when in contact with a sample suspected of containing a ligand. Will be done. These particles are any latex known or believed to be used for the aggregation of latex, such as styrene and derivatives thereof, such as methylstyrene, ethylstyrene, and chlorostyrene, olefins. , For example ethylene and propylene, acrylic acid or esters thereof, such as methyl acrylate and ethyl acrylate, methacrylic acid or derivatives thereof, such as ethyl methacrylate, acrylonitrile, and acrylamide, diene, such as butadiene, chloroprene, and isoprene, vinyl chloride. , Vinylidene chloride, and homopolymers and copolymers made from vinyl acetate. Advantageously, homopolymer or copolymer latex made from styrene, chlorostyrene, acrylic acid, vinyltoluene, methylmethacrylate is used. Other useful particles include carboxylated polystyrene, which can have a reactive group that facilitates reaction with the receptor, such as an amino group, a thiol group, a carboxyl group or another reactive group. Butadiene / styrene copolymers such as carboxylated styrene butadiene or acrylonitrile butadiene styrene are also useful. Inorganic particles such as silica, clay, carbon, such as activated carbon, and other substances on which the receptor or ligand can be immobilized can be used in the present invention. It is important that the particles are approximately the same diameter so that they can easily pass through filter openings of the same size. The particles should have an average diameter of about 0.01 to about 100 micrometers, preferably about 0.01 to about 10 micrometers. More preferably, the average diameter of the particles is about 0.3 micrometers, and the diameter of the particles does not change by more than 30% from this average, preferably less than or equal to 15%. The particles preferably have a visually recognizable color produced by the addition of dyes, pigments or coatings. For example, the preparation of pigmented polyacrylamide particles is disclosed in US Pat. No. 4,108,974 to Weghart et al., The disclosure of which is added herein by reference. The color is relatively dark, preferably black or dark blue. Preferred particles are small, uniform diameter colored polystyrenes available in various diameters from Bangs Laboratories (Carmel, CA) and Seradyn, Inc. (Indianapolis, Annapolis). Latex sphere. Treating the latex particles with a receptor corresponding to the ligand in question can be performed by any method known in the art. Treatment conditions will vary slightly as understood, depending on the physicochemical properties of the latex particles, ligands, and receptors. According to a preferred embodiment, the receptor is covalently attached to particles of defined size, preferably spherical and latex particles having a defined uniform diameter. The receptor can be an antibody, enzyme, or any protein or other substance that specifically binds to the ligand in question. The receptor is preferably an antigen or antibody. It is preferred to chemically bond the receptor to the latex particles, but the particles can be coated with the substance to which the receptor adheres, as long as the coating does not interfere with the bond between the ligand and the receptor. It is preferred to adjust the material of the receptor and particles to be immobilized so that each of the particles having a receptor aggregates when mixed together with one or more ligands for a reasonable amount of time. The sample and the particles with the receptor can be contacted in a number of ways. In a preferred method, the sample is mixed with a solution containing particles and other reagents necessary to promote the agglutination reaction to form a test mixture. Allow sufficient time intervals for agglutination to occur or otherwise for agglutination to form. Alternatively, the sample can be passed through a support containing particles and other reagents necessary to promote the agglutination reaction, such as a glass membrane. If the sample contains a ligand, aggregates and other parts will be released from the material. The released aggregates and other moieties also constitute the test mixture according to the invention. The mixture is then exposed to a filter, where the filter has openings that are larger than the particles but generally smaller than the mass of ligand / particle aggregates that may form. The filter should have a defined pore size, which is about 5 to about 15 times larger than the diameter of the latex particles, preferably about 10 to about 12 times larger, more preferably about 3 micrometers. Has a diameter of. As will be appreciated, there may be some small variation in the diameter of the holes. Preferably, the diameter of the hole does not change by more than 30% from the nominal diameter, preferably more than 15%. The pore size of the filter is chosen to retain the ligand / particle aggregates so that they still allow the passage of relatively small aggregates that may form due to non-specific aggregation. As is understood, non-specific agglutination is the agglutination of particles with a receptor in the absence of a ligand. The sensitivity of this assay should be adjusted to produce aggregates larger than the pore size, approximately 10-15 particles in diameter. Preferably, the filter will be a full channel membrane with controlled diameter pores. Preferred controlled pore membranes are those made of polycarbonate, such as those commercially available from Poletics Corporation (Livermore, Calif.). Once the mixture is filtered, the resulting filtrate is analyzed for the presence of non-aggregating or non-specifically agglomerating particles. As will be appreciated, such analysis can be performed by suitable physical and / or chemical methods known in the art, such as centrifugation or particle counting, but the filtrate is preferably filtrate. This is done by visual inspection to determine the presence in it of a recognizable color that corresponds to the latex particles. Thus, when the ratio of the particle with the receptor to the ligand was carefully selected, a qualitative system was established, where the presence in the filtrate of the color corresponding to the particle indicates the absence of the ligand of the sample. And the absence of such a color in the filtrate indicates the presence of the ligand in the sample. Also, of course, the abundance of the ligand in the sample according to the present invention can be determined. A suitable quantitative system can be established by comparing the filtrate with one or more visual standards that correspond to a known concentration of colored particles in the filtrate. Such visual standards will be prepared from samples with known concentrations of ligand. The present invention also provides suitable equipment for performing the methods described for ligand assays. In general, such devices are filter means that form a mixture by contacting a sample suspected of containing a ligand with particles having a receptor, the particles having the ability to form aggregates when in contact with the ligand. The filter means has an opening larger than the particles but smaller than the agglomerates; and an analytical means for determining the presence of the particles in the filtrate. A preferred apparatus for carrying out the method of the present invention is the assay plate (1), examples of which are shown in FIGS. 1-4. Assay plates of the invention generally have an upper member (10) having a filter well (12) and an observation well (14); a filter means (20) that is adjacent to and extends across the filter well; the filter means. Consists of a core means (30); adjacent to and extending the length of the filter well and the observation well; and a bottom means (50); adjacent to the core means. As will be understood, analytical means consist of assay plate elements other than filter means. The superstructure is preferably made of a material that is substantially impermeable to aqueous solutions, such as aqueous solutions associated with the human body. The superstructure can preferably be cut or punched out of the rigid material, thus providing some support to the assay plate. The superstructure is made of polystyrene and preferably has a length of about 100 mm, a width of about 20 mm, and a thickness of about 1.0 mm. The superstructure should be cut, punched, or otherwise made to have filter wells (12) and observation wells (14) that extend throughout its thickness. Preferably, the filter wells and observation wells are circular, but other shapes are possible. Also, the filter well and the observation well preferably have a predetermined distance (X) from each other. A previously determined distance (X) is the aggregation of ligand / particles passing through the filter, as some ligand / particle aggregates may not form a mass of sufficient diameter to be retained by the filter. The object is selected so that it does not reach the observation window. Thus, the previously determined distance (X) will vary with the particular ligand, receptor, particles, and core means used. In general, it would be the case when the distance (X) changes in the opposite direction to the ability of the core means to hold the agglomerates. The filter means (20) is preferably a filter as described above having an opening (22) that is larger than the particles but generally smaller than the mass of ligand / particle aggregates. The filter is preferably a polycarbonate film with controlled pores. The filtering means only needs to extend across the filter wells, but if the filtering means is transparent or almost transparent, for example if the filtering means is a polycarbonate membrane with controlled pores, the filtering means is also preferably. , Extend across the observation well, as shown in FIG. There is a core means (30) adjacent to the filter means. Preferably, the wick means is located in close physical contact with the filter means, thus the filtrate flows vertically into the wick means and moves horizontally from below the filter well to below the observation well. To do. The filter means only needs to extend the length of the filter well and the observation well, but the core means is preferably slightly longer, as in FIG. The filter and core means are preferably attached to each other with a porous adhesive, for example an adhesive available from the Adhesive Research Company (Glen Rock, PA) under the trademark ARcare Porous. The core means is preferably made of glass or a natural or synthetic polymeric material, such as polyester non-woven fibers. The composition and arrangement of the fibers in the core means is chosen so that the agglomerates and particles move over them at different rates. Preferably, the particles move faster. The core means also has a visually recognizable pattern, eg, a cross-hatch pattern (32), embossed or otherwise formed to facilitate visual detection of color in the observation well. Is preferable. The bottom means (50) consists of a material that is adjacent to the core means and is preferably impermeable to aqueous solutions. The bottom means is preferably cut or punched to approximate the width and length of the top member. The top member and / or bottom means serve to support the assay plate. Thus, if the top member provides adequate support, the bottom means can be made of a relatively rigid material, such as a vinyl polymer. The floor means is preferably physically attached to the other components of the assay plate with an adhesive. A preferred assay plate is a barrier (40), eg, a bottom means having at least the length of the core means and preferably having the core means and the adhesive, as a large number of suitable adhesives impair the suction properties of the core means. It has a thin polyethylene film located between. The assay plates of the present invention also include a support (60), eg, a glass membrane, containing particles with receptors and other reagents necessary to promote the agglutination reaction, if desired. Such supports should be used if the sample should be applied directly into the filter wells rather than premixed with a solution containing particles with receptors. As shown in FIG. 4, the support should be located between the top member and the filter means and extend across the filter well. The present invention further provides a reaction cell for use when contacting a sample with particles having a receptor. The preferred type of reaction cell (70) is depicted in FIG. One element of the reaction cell according to the invention is a container (80) in which particles with receptors can be contacted with a sample suspected of containing a ligand. Such containers can have various shapes. However, the preferred shape of the container is that of a pipette, eg, that shown in FIG. As will be appreciated, the container with the open end preferably consists of a cap (72) for containing the sample and the particles with the receptor. Preferred containers are disposable and consist of any relatively inexpensive, substantially transparent synthetic polymer known in the art. Suitable clear pipette-shaped containers are available from Franklin, Inc. (Franklin, NJ). A preferred reaction cell is a destructible vessel (90) containing particles (92) with receptors. The destructible container can consist of glass or some synthetic polymer and of contact, as long as the material uses sufficient structural integrity to contain the particles tightly until the particles should be in contact with the sample. When the container is destroyed or ruptured by the applied force. If the reaction cell contains a destructible vessel, it is necessary to construct the vessel from a flexible material through which such bursting force can be applied to the destructible vessel. Preferred reaction cells further contain a killing solution. The term "killing solution" means a solution that has the ability to biologically inactivate a component used when performing a ligand assay-eg, a ligand, receptor or sample. Solutions consisting of ethanol, formaldehyde, glutaraldehyde, iodophore, or oxidative bleach provide examples of killing solutions according to the present invention. The killing solution preferably contains an oxidizing bleach, for example sodium hypochlorite. Preferably, the killing solution is contained in a compartment (100), which is located at one end of the container and separated from it by a rupturable membrane (102). Alternatively, the killing solution is contained in a burstable container (110) located within the container. This membrane or destructible container consists of a membrane that has sufficient structural integrity to tightly contain the killing solution until it is destroyed or ruptured by the applied force. The killing solution is then usually released when the assay is complete and contacted with a biologically active substance located in the vessel or on the assay plate. The present invention also provides kits useful for ligand assays. Some of these kits consist essentially of the aforementioned receptor-bearing particles and filter means. The other kit consists of an assay plate and a reaction cell. Additional objectives, advantages and novel features of the present invention will become apparent to those skilled in the art upon examination of the following examples. The following examples are not intended to be limiting. Unless otherwise stated, parts and percentages are by weight. Example 1 Purified HIV-1 antigenic protein in 0.1 mol glycine-buffered saline (GBS) solution (pH = 8.0) into pigmented polystyrene latex particles with an average diameter of approximately 0.3 micrometers. It was passively adsorbed at 4 ° C for about 12 hours. The particles were then centrifuged and washed with GBS solution containing 0.1% bovine serum albumin (BSA). The particles are then added to the GBS solution, along with certain other components believed to optimize the rate and sensitivity of the agglutination reaction, to produce a solution consisting of the following components: particles (0.5%); BSA (0.1%); polyethylene glycol (6%); polyvinylpyrrolidone (1%); sodium azide (0.5%) and free HIV-1 antigenic protein (1.0 picogram / ml). Example 2 Purified antigenic protein isolated from Borrelia burgdorferi culture in 0.1 mol phosphate buffer (PBS) solution (pH = 7.2) with an average diameter of approximately 0.3 micrometer. It was passively adsorbed on dyed polystyrene latex particles having a pH for about 12 hours at 4 ° C. The particles were then centrifuged and washed with PBS solution containing 0.1% BSA. The particles are then added to the PBS solution, along with some other component believed to optimize the rate and sensitivity of the aggregation reaction, to produce a solution consisting of the following components: BSA (0.2%); Particles (0.5%); Polyethylene glycol (6%); Ethylenediaminetetraacetic acid (EDTA) (1%); and Sodium azide (0.5%). Example 3 Purified antigenic protein isolated from cytomegalovirus culture is passively transferred to dyed polystyrene latex particles with an average diameter of approximately 0.3 micrometers in 0.1 mol GBS solution (pH = 7.0). It was adsorbed at 4 ° C for about 12 hours. The particles are then added to the GBS solution, along with some other component believed to optimize the rate and sensitivity of the aggregation reaction, to produce a solution consisting of the following components: particles (0.5%); BSA (0.1%); polyethylene glycol (6%); sodium chloride (1.5 mol); and sodium azide (0.5%). Example 4 A 100 μl sample of human serum known to contain the HIV-1 virus was placed in a pipette-shaped reaction cell, eg, the reaction cell shown in FIG. The destructible vessel in the reaction cell contained 200 μl of the solution prepared in Example 1. The reaction cell was closed by replacing its cap and ruptured by squeezing the destructible ease between the thumb and index finger. After about 10-30 seconds, the cap was removed and a few drops of the dark blue sample / particle mixture were placed in the assay plate, eg, the filter well of the assay plate shown in FIG. The assay plate had a polycarbonate membrane with controlled pores of approximately 3 micrometers and a polyester suction layer. The distance (X) between the filter well and the observation well was 0.25 inches. After about 1-3 minutes, a dark blue mass of particle / ligand aggregates was observed in the filter wells. No dark blue color was observed in the observation wells. As will be appreciated, when a reaction cell as shown in FIG. 6 or 7 is used in place of the reaction cell of FIG. 5, the membrane (102) or destructible vessel (110) is destroyed. Alternatively, the biologically active substance can then be inactivated by contacting the ruptured and released killing solution with the vessel and / or assay plate. Example 5 The procedure of Example 4 was repeated, but serum samples known to be free of antibodies to the HIV-1 virus were used. After about 1 minute, only a few clumps of particle / ligand aggregates were observed in the filter wells. The dark blue color was observed in the core material under the observation well. Example 6 The procedure of Example 4 was repeated, except that human serum samples containing an antibody against Borrelia burgdorferi instead of an antibody against the HIV-1 virus were used. The solution from Example 2 was used in place of the solution from Example 1. After about 1-3 minutes, a dark blue mass of particle / ligand aggregates was observed in the filter wells. No dark blue color was observed in the observation wells. Example 7 The procedure of Example 6 was repeated, but serum samples known to contain no antibody against Borrelia burgdorferi were used. After about 1 minute, only a few clumps of particle / ligand aggregates were observed in the filter wells. The dark blue color was observed in the core material under the observation well. Example 8 The procedure of Example 4 was repeated, except that human serum samples containing antibodies against cytomegalovirus were used instead of antibodies against HIV-1 virus. The solution from Example 3 was used in place of the solution from Example 1. After about 1-3 minutes, a dark blue mass of particle / ligand aggregates was observed in the filter wells. No dark blue color was observed in the observation wells. Example 9 The procedure of Example 8 was repeated, but serum samples known to contain no antibody against cytomegalovirus were used. After about 1 minute, only a few clumps of particle / ligand aggregates were observed in the filter wells. The dark blue color was observed in the core material under the observation well. Example 10 A 200 μl sample of human serum known to contain antibodies against the HIV-1 virus was pipette into the filter wells of an assay plate as shown in FIG. The assay plate had a glass membrane support (60) saturated with the solution prepared in Example 1. The assay plate had a controlled pore of about 3 micrometers and a polyester suction layer. The distance (X) between the filter well and the observation well was 0.25 inches. After about 1-3 minutes, a dark blue mass of particle / ligand aggregates was observed in the filter wells. No dark blue color was observed in the observation wells. Example 11 The procedure of Example 10 was repeated, but serum samples known to be free of antibodies to cytomegalovirus were used. After about 1 minute, only a few clumps of particle / ligand aggregates were observed in the filter wells. The dark blue color was observed in the core material under the observation well.