JPH0449911B2 - - Google Patents

Description

Claim 1: A reaction layer containing a detectable species and holding a predetermined amount of a diffusible reagent adapted to be substantially non-diffusible by the analyte as a function of the concentration of the analyte; a registration layer suitable for receiving a reagent; an insulating layer between the reaction layer and the registration layer, into which the diffusible reagent can permeate; and wherein the diffusible reagent is measured. a multilayer element for the analysis of liquids containing an analyte, characterized in that the insulating layer prevents detection of the detectable species retained in the reactive layer, the insulating layer being present in an amount in excess of the amount that is reactive with the analyte of interest; . 2. The element of claim 1, comprising a sample spread layer for receiving the liquid to be analyzed. 3. The element of claim 2, wherein the spread layer is an isotropic non-fibrous spread layer. 4. The element of claim 2, wherein said spread layer is fibrous. 5. The element of claim 1, wherein the detectable species is a fluorescent substance. 6. The element of claim 1, wherein the detectable species is a radioactive substance. 7. The element of claim 1, wherein the insulating layer is opaque to radiation from the detectable species. 8. The element of claim 1, wherein said liquid contains an antigen (antibody) and said reactive layer contains a diffusible antibody (antigen) having bound thereto a detectable species. 9. The element of claim 2, wherein said spread layer is a fabric. 10. The element of claim 2, wherein the reaction layer comprises agarose. 11. The element of claim 1, wherein the insulating layer comprises titanium dioxide. 12. The element of claim 1, wherein said registration layer comprises agarose. 13. The element of claim 1, wherein said registration layer comprises a polymer emulsion. 14. The element of claim 1 comprising a radiolucent substrate carrying each of said layers. 15. The element of claim 14, wherein said substrate is polystyrene. 16. Adding an analyte-containing liquid to an element comprising multiple layers, including a reaction layer and a registration layer containing a predetermined amount of a diffusible reagent containing a detectable species, and causing the analyte-containing liquid to diffuse into said registration layer. determining the amount of reagent, and wherein the diffusible reagent is present in an amount in excess of the amount that is reactive with the analyte to be measured and is non-diffusible by the analyte as a function of the concentration of the analyte. A method for analyzing an analyte in a liquid, characterized in that the registration layer is suitable for receiving reagents diffusing into it. BACKGROUND OF THE INVENTION Numerous devices are known for the chemical analysis of substances in liquids, especially biological fluids. Some types of devices require expensive equipment, highly trained operators, and large volumes of analytical samples. Other types of devices are relatively simple, portable, multilayer elements that perform a single test at a time without expensive equipment. US Patent No. 23, 1973
No. 3,723,064 is directed to a layered test device comprising a layer comprising a permeable member impregnated with a chemical reagent for reaction with a test substance and having a first region and a second region of different permeability to a test fluid. It is being The permeable layer is disposed adjacent to the permeable member and reacts with the reaction products resulting from the reaction of the chemical reagent with the test substance to produce a visible reaction indicative of the presence and concentration of the test substance. to help draw out the next layer, which is an indicator layer impregnated with suitable chemicals to produce a . U.S. Patent No. 16 November 1976
No. 3,992,158 discloses an integrated analytical element that includes a spread layer and a reagent layer. The spread layer is an isotropic non-fibrous material that spreads at least one component of the applied liquid sample within itself to obtain a uniform concentration, which concentration then transfers to an adjacent reagent layer where it can react to form a detectable species. It is a porous layer. U.S. Patent No. 16 August 1977
No. 4,042,335 is directed to a multilayer element for the analysis of liquids such as biochemical and biological fluids.
The element includes a reagent layer containing a composition that provides a diffusible, detectable species that is interactive with the substance to be analyzed, and a registration layer that is permeable to the detectable species, within which the detectable species is permeable. species can be detected. The element may also include a spread layer and a radiation blocking layer to enhance detection of detectable species. US Patent No. 27, 1977
No. 4,050,898 discloses a spread layer of at least two superimposed layers and a reagent, wherein the spread layer is a non-fibrous isotropic porous spread layer containing a nonionic surfactant that normalizes the transport of the spread layer. Contains layers. U.S. Patent No. 3, January 3, 1976
No. 4,066,403 includes a first reagent that reacts with an analyte to produce a degradation product and a second reagent that is suitable for reacting with the degradation product to produce a detectable change, wherein the interfering substance is substantially A barrier to said decomposition products, impermeable to said components, is directed towards said integrated analytical element for analysis of a complex fluid disposed between said first reagent and said second reagent. US Patent No. 17, 1978
No. 4,069,016 discloses contacting an aqueous liquid sample with a diffusible bilirubin-displaceable detectable ligand bound to a carrier capable of also binding bilirubin, and then contacting the so-displaced detectable ligand. The present invention is directed to a method for quantifying bilirubin in an aqueous liquid sample, the method comprising: detecting bilirubin in an aqueous liquid sample; US Patent No. 13, 1979
No. 4,144,306 discloses an interactive composition in which a reagent layer includes a non-diffusible material having a preformed detectable moiety bonded to the non-diffusible material, the composition comprising a liquid containing an analyte. Directed to a displacement/release method using a multilayer analytical element comprising at least a reagent layer and a registration layer, yielding a diffusible product comprising a preformed detectable moiety upon displacement by an analyte in the presence . The registration layer receives the diffusible product. Other layers may also be used, including supports, spread layers, radiation blocking layers, etc. Examples of such interactive compositions include labeled antigen-antibody complexes that displace the labeled member from which the analyte migrates to the registration layer. U.S. Patent No. 28 August 1979
No. 4,166,093 discloses a radiation-transparent reagent layer containing a composition capable of interacting with an analyte and providing a radiometrically detectable species, an analyte-permeable porous radiation-blocking layer, and between the reagent layer and the radiation-blocking layer. an intervening radiolucent detectable species migration inhibition layer, the detectable species migration inhibition layer being permeable to the analyte and radiometrically detectable when the element contacts the liquid under analysis; It is directed to a liquid analytical element that inhibits migration of species into the porous radiation blocking layer. US Patent No. 24, 1981
No. 4,258,001 is directed to a multilayer element for competitive immunoassays that includes a zone with a particulate structure that includes heat-stable, non-swellable organic polymer particles and an adhesive. Immunological reagents such as fluorescently labeled antigens can be attached to the particles. In Example 50, a competitive immunoassay element is described in which antibodies are immobilized in a spread/reagent zone dispersion and labeled antigen is premixed prior to application to the test element. It is also stated that labeled antigens can be incorporated into the element. However, it is stated that if labeled antigen is incorporated into the element, care must be taken to keep the labeled antigen separate from the immobilized antibodies in the element to avoid premature binding of labeled antigen and antibody. (Column 30, lines 2 to 7). Immunochemistry 3rd edition by DMWeir, Blackwell.
Blackwell Scientific Publication, Oxford, UK, Volume 1, Chapter 19 describes a radial quantitative immunoprecipitation technique in which an antigen-containing specimen is placed in the center of a colloidal layer containing antibodies. is listed. The distance that the antigen radiates from the center is measured to give a quantification of the antigen in the specimen. European Patent Application No. 0051183 filed October 15, 1981 states that antigens (or antibodies)
A multilayer analytical element comprising a porous medium layer in which is immobilized and a layer in which a substance that does not participate in immunoprecipitation in the porous medium layer can diffuse, and after which the amount of the substance can be determined using a detection means. is disclosed. In operation, the antigen (or antibody) to be analyzed is labeled and then applied to the porous media layer. The labeled antigen (or antibody) reacts with the immobilized antigen (or antibody) to form a complex. Excess unreacted antigen (or antibody) is essentially separated from the complex and then diffuses into the reagent layer. Unreacted labeled antigen (or antibody) is measured optically to provide a quantification. This patent application presents the quantitative nature of gel-mediated precipitation reactions. SUMMARY OF THE INVENTION A reaction layer containing a detectable species and a predetermined amount of a diffusible reagent suitable to be substantially non-diffusible by said analyte as a function of the concentration of said analyte; a registration layer adapted to receive a diffusing reagent; a means for detecting the reagent in the registration layer; and a means for discriminating between detectable species in the reagent layer and the registration layer. A multilayer element for the analysis of liquids containing an analyte, characterized in that it comprises: an insulating layer between said reaction layer and said registration layer suitable for providing analyte-containing liquids.

[Brief explanation of the drawing]

FIG. 1 is a schematic cross-sectional view of a preferred multilayer element of the invention; FIG. 2 is a schematic cross-sectional view of the element of FIG. 1 showing the location of some of the components of the element prior to contact with the analyte; FIG. 3 is a schematic cross-sectional view of the element of FIG. 2 showing the location of some of the components of the element after contact with a specimen. DETAILED DESCRIPTION OF THE INVENTION The present invention contemplates an analytical technique that includes a predetermined concentration of reagent in an analytical element suitable for reacting with an analyte. That is, the analyte reacts with an amount of reagent that is normally diffusible relative to the amount of analyte and then immobilizes that amount. The unreacted reagent, which also contains the detectable species, diffuses into the registration layer, where the amount of reagent in the registration layer is determined by the detectable species using a suitable detection means. . Preferably, an insulating layer is used between the reaction layer and the registration layer to limit detection of the detectable species to those diffused into the registration layer. Although the present invention is suitable for the analysis of virtually any substance that can be reacted with a second diffusible substance having a detectable species to immobilize said second substance, the present invention does not require immunoprecipitation techniques. It is particularly suitable for applications using For simplicity, the invention will be described in terms of immunoprecipitation. Analytical techniques can be classified as heterogeneous or homogeneous methods. Heterogeneous methods require physical separation of free and bound reactants but are believed to be more sensitive. Homogeneous methods are preferred due to their ease and simplicity of operation, but suffer from high background levels caused by incomplete discrimination between bound and free species. In homogeneous methods, no physical separation occurs, but rather one modulation of the reactant label based on the state of complexation of the carrier. The reduced level of sensitivity is due, at least in part, to incomplete discrimination. The present invention uses a heterogeneous immunoprecipitation technique in an integrated, one-step format, thus obtaining the operational simplicity of a homogeneous technique while having the sensitivity of a heterogeneous technique in a thin film format. The novel elements and operations of the present invention provide separation of bound and unbound species using immunoprecipitation techniques in a single step and in preformed thin film format, with detection dependent on differential diffusion of detectably labeled antibodies. This is done in a manner that avoids competitive reactions with additional antigenic species. Thus, according to the present invention, any substance that produces immune complexes can be analyzed without the use of reactants other than those that produce immune complexes. As used herein, the term "immune complex" refers to any combination of antibody-rich to antigen-rich complexes, either soluble or insoluble, including immunoglobin and haptenic or multivalent antigens and heterogeneous or monoclonal antibodies. It is intended to represent the percentage of aggregates of said immunoglobin with the combined ligand. During the operation, a predetermined volume of sample liquid containing the analyte, a measured amount of diffusible antibody placed in a pre-formed thin film (where the diffusible antibody contains a detectable species bound to this antibody) be contacted. The antigen analyte associates with the antibody to form a complex species that is substantially less diffusible than uncomplexed antibody, quantitatively proportional to the concentration of antigen in the sample fluid. Separation of complexed and uncomplexed antibodies occurs due to differences in the diffusion rates of the uncomplexed antibody in excess of that required to form a complex with the antigen. Determination of the amount of uncomplexed antibody that initially diffuses out of the layer in which the antibody is placed and, preferably, the registration layer, where the antibody is concentrated by immobilization and is concentrated in the element. This is done by measuring the amount of antibody that diffuses (preventing any back-diffusion or migration of the antibody). The measurement is carried out using antibody-
This is done by measuring the amount of antigen complex species and therefore the amount of detectable species in the registration layer which is indicative of the concentration of antigen analyte in the sample liquid. Although the present invention is primarily described for the analysis of antigen using a diffusible antibody with a detectable species bound to the diffusible antibody, since immunoprecipitation techniques are used, It is to be understood that antibodies can be assayed using diffusible antigens that have detectable species. For simplicity, the invention has been described in terms of antigen as the analyte, but it should be understood that the invention encompasses both operations. The following example illustrates the principle of thin film immunoprecipitation. Example 1 3 mil polystyrene base material [Dow Chemical
Sold under the trademark TRYCITE by Company, Midland, Michigan]
The following materials were applied on top. 10 mg/ft ² Trademark Immunoglobulin (FITC-Anti-Human Albumin, sold by Atlantic Antibodies, Inc., Scarborough, Maine);
1000 mg/ft ² agarose [SEAKEM LE, sold by F.M.C. Marine Colloids, Rotkland, Maine] and 0.1 mg/ft2
ft ² surfactant [p-nonylphenoxy polyglycidol sold under the trade name OLIN 10G by Olin Chemical Corporation, Stanford, CT].
The substrate thus coated is dried and then 10 cm
Cut into 1 cm x 1 cm strips. This strip is one of the strips.
The antigen (globulin-free human albumin, sold by Sigma Chemical Company, St. Louis, Missouri) was prepared by soaking the sample in a solution of the antigen for 10 minutes, then rinsing in distilled water for 10 minutes, and then air drying at room temperature. ) was exposed to. 0 mg/ml 0.5 in phosphate buffered saline
An antigen concentration series consisting of mg/ml, 1 mg/ml and 2 mg/ml was prepared. The relative fluorescence intensity of the sample is the excitation
450nm, slit 5nm, emission scanning 470nm to 600n
Measurements were made from exposed and unexposed portions of individual sample strips using a FLOUROLOG with m and integrated photon counting mode (Specx Industries, Inc., Metatitanium, New Jersey). Comparisons between samples were made by calculating the % decrease in fluorescence caused by exposure to antigen solution. Referring to Table 1 below, it can be seen that higher levels of antigen immobilize higher levels of fluorescent antibody. Table 1 Sample No. [Ag] (mg/ml) Fluorescence intensity (%) 1 0 29.5 2 0.125 26.5 3 0.250 23.0 4 0.375 21.2 5 0.500 18.5 It reacts effectively and measurably with the pre-coated antibody, yet diffusion of the antibody out of the layer occurs differentially and inversely proportional to the amount of antigen added to the layer, thus controlling the mechanism of antigen quantification. It shows giving. The novel elements of the invention include, in its simplest form, a sample spread layer; a reaction layer comprising a diffusible antibody having a detectable species attached to the diffusible antibody; a substantially radiopaque porous layer; a registration layer suitable to receive and substantially immobilize the antibody that diffuses into the registration layer; and a radiation transparent substrate supporting each of the layers. The spread layer has a porosity that retains a constant volume per unit area of liquid after completion of sample absorption and provides a precise supply of a measured amount of sample liquid to the reaction layer, thus allowing for dispensing or measurement. No equipment is required. As an example of a material suitable for use as a spread layer, a fibrous layer with a special predetermined porosity, U.S. Pat. No. 3,992,158
Isotropic porous non-fibrous materials disclosed in US Pat.
The fabric of No. 4292272 can be mentioned. In another embodiment, the spread layer includes, for example:
It may also include a fibrous coating of organic or inorganic fibers such as paper, asbestos or potassium titanate. The reaction layer includes a hydrophilic colloid that is liquid sample permeable, inert to proteins, swellable but insoluble to maintain wet strength integrity, and yet binds to antibodies. An antibody having a detectable species is first placed and is non-reactive and non-binding to said antibody.
Suitable materials for the reaction layer include gelatin derivatives,
Hydrophilic, which includes both naturally occurring substances such as polysaccharides such as hydrophilic cellulose derivatives, dextran, gum arabic, and agarose, and synthetic substances such as water-soluble polyvinyl compounds such as polyvinyl alcohol and polyvinylpyrrolidone, acrylamide polymers, etc. There are materials that can be gelled. Thermoreversible gels are preferred due to their ease of fabrication. The polymer used as the reaction layer may be crosslinked if desired, but care must be taken to avoid deactivation of the antibody by the crosslinking agent. Detectable species may include conventional labeling means known in the art. Among suitable detectable species, mention may be made of radioactive labels such as gamma detection, fluorescent labels, enzymatic labels (giving a colorimetric signal) and luminescent labels. In a preferred embodiment, the fluorescent material is
In particular, it is used as a detectable species because of its detection sensitivity. In a preferred embodiment, the hydrophilic colloid of the reaction layer also contains a permeate that enhances the diffusion constant of the labeled antibody in the polymer matrix, i.e., the permeate effectively increases the pore size of the hydrophilic colloid medium. . Preferably, the permeate is a hydrophilic non-gelling polymer such as polyvinylpyrrolidone or sorbitol. Without wishing to be bound by theory, it is believed that the permeate reduces the degree of hydrogen bonding of the gelling polymer, providing less resistance to diffusion of the antibody by the detectable species. When using fluorescence detection techniques, care should be taken to avoid the use of substances that can reduce the quantum efficiency of the fluorescence and thus make the antibody substantially less detectable. To illustrate the effectiveness of polymer matrix permeability, comparative radial diffusion measurements were performed on various coated polymer matrices. Example 2 An agalome (Sechem LE) solution was prepared with the permeate additive described below and then treated with an adhesive polymer [RHOPLEX, Rohm and Haas Company, Philadelphia, Pennsylvania] to promote adhesion. Location] 68.3g, 10% solution of dioctyl malonate 5.6
g, 26.7 g of a 10% colloidal silica dispersion [LUDOX, E.I. Dupont de Nemois Company, Wilmington, DE], 40 g of a 10% solution of phthalated gelatin;
Polynonyl phenoxyglycidol (Oline
A composition containing 3 g of a 50% solution of 10G) and 862 g of water was coated by extrusion onto a primed polystyrene substrate (TRYCITE). A 5% solution of fluorescein sodium salt was used as the tracer dye. The coated sample to be tested is attached to a glass plate by means of double-sided adhesive tape and then attached to a Selectrasol
Testing was conducted on the TLC-Solvent Selector System (Schleicher & Schell, Inc., Keene, New Hampshire). The tracer dye solution was applied to the center of each test sample in a humid environment through a porous core to prevent premature evaporation of the solvent. Table 2 summarizes the results.

[Table] The carbo wax used has a molecular weight of 3000~
37,000 science and technology companies
Polyethylene Glycol Co., Ltd., Fairhaven, New Jersey. The polysucrose was FICOLL 400, sold by Pharmacos Fine Chemicals, Atpsala, Sweden. Table 2 illustrates the effectiveness of various permeants in increasing the rate of diffusion through an agarose layer. The porous opaque layer is permeable to diffuse antibodies carrying the detectable species, yet provides opacity that eliminates any possibility of detection of the detectable species other than in the recording layer. Prevent reading. In other words, the porous opaque layer prevents detection of the detectable species retained in the reaction layer. For example, in the case of fluorescently detectable species, the porous opaque layer absorbs in the excitation radiation, eg in the ultraviolet range, while maintaining reflectivity in the fluorescent radiation range, eg in the visible range. Thus, while the excitation radiation is effectively suppressed from reaching the bound label, the emitted radiation from the registration layer is reflected rather than absorbed. The porous opaque layer may include a suitable reflective material in the binder. Preferably, the titanium dioxide in the agarose is coated with varying coverage to provide the desired porosity. If desired, the porous opaque layer may also contain suitable additives to improve opacity and reflectance. Since many pigments have high surface binding coefficients for nonspecific absorption of immunoglobulins, such pigments can be compared with other pigments that are strongly adsorbed to the surface of the pigment but have no affinity per se for the immunoreagent. It is desirable to pre-treat with a polymer. The examples below illustrate the effectiveness of surface treatments to prevent unwanted immunoglobulin binding to particulate materials. Example 3 Surface Treatment Method 100 g of titanium dioxide (TiPure R-931, E.I. Dupont Denemos Company, Wilmington, DE) was injected into photographic grade gelatin mold 9168 [Rousselot Corp., Paris, France]. location] 1
% solution and then stirred at 40°C for 1 hour. At the end of this time, the titanium dioxide was separated by low speed centrifugation. Excess gelatin was removed and the titanium dioxide was resuspended in distilled water and then centrifuged. This gelatin removal step was repeated three times. Example 4 Determination of surface activity Pigment treated according to the procedure of Example 3 (0.3 g)
Samples were stirred for 1 hour with 3.0 g of labeled immunoglobulin (FITC-anti-human albumin, Atlantic Antibodies, Inc., Scarborough, ME) and diluted 1:9 with phosphate-buffered saline. Then, it was diluted with water to a total amount of 6.89 g. Add this mixture to AMICON XM-3000.
An ultrafiltration cell with a membrane filter (Amicon, Inc., Cambridge, Mass.) was filled and then centrifuged at low speed for several minutes. 1 ml of ultrafiltrate was diluted 1:9 with phosphate buffered saline and then assembled into a Beckman 5864 Spectrophosmeter Fluorescence Accessory Model 196776 for fluorescence reading. (Beckman Instruments, Inc., Fullerton, Calif.) to measure fluorescence intensity. The target sample was an immunoglobulin solution at the same dilution as the test sample, and was also ultracentrifuged like the test sample. Table 3 contains fluorescence intensity (FI) measurements from the samples normalized to 100% for the control. Table 3 Samples FI Untreated TiO ₂ 4.4 Treated TiO ₂ (Example 3) 54.4 Control (Immunoglobulin Solution) 100.00 Table 3 illustrates the effectiveness of pigment treatments in blocking immunoglobulin binding. The registration layer serves as a location for concentrating the diffusing antibodies by immobilizing them with a detectable species. Detection means are used for determining the amount of detectable species in this layer. Materials suitable for non-specifically binding, immobilizing or "mordanting" antibodies are known in the art. For example, the Journal of the
Reticuloendothelial Society) Volume 3, Page 29~
See page 40 (1966). Although many of the materials known in the art to immobilize or mordant antibodies work in solution, these materials are inactivated in a gelling environment such as agarose and thus used in the present invention. seems to require special care in its selection. The following non-limiting example illustrates the effectiveness of non-specific adsorption properties in a registration layer for immobilization of labeled immunoglobulin. Example 5 A series of coatings were prepared by using agarose as a gelling environment with the following materials incorporated therein as a discrete phase. A surfactant, p-nonylphenoxy polyglycidol (Olin 10G), was added as a coating aid. Coatings were evaluated by contacting each coating with a fixed volume of labeled antibody solution for exactly 10 minutes. The antibody solution used was the same as shown in Example 3. At the end of the contact time, the coating was rinsed with distilled water and then air dried at ambient temperature. Fluorescence (arbitrary relative units)
was determined by the same technique as described in Example 4.

[Table] Polyvinylpyrrolidone

[Table] Minium

Polymer A consists of 4-vinylpyridine (4VP) and benzyltrimethylammonium chloride (TMQ) on hydroxyethylcellulose (HEC).
The graft ratio was 2.2HEC/2.24VP/1TMQ. The starch was a soluble starch sold by Mallinckrodt, Inc., Paris, Kentucky.
The polyethylene oxide was an emulsion sold under the trade name POLYOX WSR-N-80 by Union Carbide Corporation, New York, New York. Titanium dioxide is Example 3
It was the same as that described in . The polyvinylpyrrolidone was type NP-X15, sold by GAF Corporation, New York, New York. Aluminum oxide (0.3 micrometer) was purchased from Fluka Chemical Co., New York.
Sold by Hauppauge location. Alternatively, the antibody can be pre-coated in the registration layer to impart mordant properties. See, eg, the aforementioned US Pat. No. 4,258,001. However, this does not require the use of the registration layers listed in Table 4. This is because these registration layers are not only effective mordants for antibodies;
This is because it is also non-specific. That is, these registration layers bind substantially all immunoglobulin G regardless of its source. The radiolucent substrate used in the present invention is transparent to the radiation emitted by the detector and the detectable species. For example, in preferred embodiments, the detector emits excitation radiation and detects fluorescence emitted by the detectable species.
Examples of suitable film substrates for use as radiation transparent substrates include materials such as cellulose acetate, polyethylene terephthalate, polycarbonate and polyvinyl compounds such as polystyrene. To promote adhesion,
Preferably, the film substrate is primed. It should also be understood that the novel analytical elements of the present invention may contain optional layers such as filter layers, timing layers, etc. For example, the filter layer is
It can be used on the spread or reaction layer to remove small molecules and particulates that may interfere with the analysis. Additionally, a timing layer may be provided to adjust the delay between contact of the antibody with the reactive layer and diffusion of the antibody with the detectable species, such that the diffusion of the antibody occurs before the antigen has a chance to bind to the antibody in the reactive layer. It would also be desirable to avoid any resulting erroneous readings. A filter layer can also be used to prevent foreign substances from entering the registration layer. Referring to the drawings, FIG. 1 is a cross-sectional view of a preferred element of the invention. Element 10 consists of the following layers in order: i.e. a spread layer 12 to which the analyte is applied; a reactive layer containing antibodies with detectable species;
14; timing layer and/or as above
an intermediate layer 16 which may contain a filter layer; a registration layer 20 containing a concentration or immobilization element for the diffusing antibody with the detectable species to detect remaining in the reaction layer; a porous opaque layer 18 suitable for isolating the species; It consists of a radiation transparent layer 22. 2 and 3 illustrate the operation of element 10 of FIG. A sample fluid containing an analyte, in this case an antigen, the substance under analysis, is applied to the spread layer 12 which receives a uniform, predetermined amount and then diffuses into the reaction layer 14. Antigen is layer 14
to form a substantially non-diffusion complex. Antibodies in excess of antigen, which are normally able to diffuse through the sample liquid element, diffuse through intermediate layer 16 and porous opaque layer 18 and are immobilized in the registration layer. Then, the detectable species is activated by incident radiation passing through the radiation transparent layer 22, and the amount of the detectable species is then determined. The following non-limiting examples illustrate the operation of elements of the invention. Example 6 10 ml of 0.1% human serum albumin (antigen) (Sigma Chemical Company, St. Louis, MO) and fluorescein isothiocyanate (antibody) (Atlantik Antibodies, Inc., Scarborough, ME). Goat anti-human serum albumin IgG fraction labeled with 1:5 diluted in phosphate-buffered saline.
100 ml were mixed in the test. A whitish precipitate formed immediately. The contents of the test tube were (a) agarose 1000 mg/ft ² and molecular weight 3000 ~
3700 (Fitzsier Chemical Company,
(b) titanium dioxide pretreated with gelatin (E.I. Dupont de Nemois, Inc., Wilmington, DE ⁾ ; (b) titanium dioxide pretreated with gelatin; sold under the trade name TiPure R-933) 520 mg/ft ² and agarose 500 mg/ft ²
(c) a registration layer comprising 1000 mg/ft ² of agarose and 100 mg/ft ² of polyethylene glycol molecular weight 4000; and (d) a 3 mil primed polystyrene substrate. (Dow Chemical Company, Midland, Michigan). After a 2 hour absorption period, UV light was shined through the base of the element and human serum albumin through a control element that had not been coated with goat anti-human serum albumin. For controls, a significant amount of fluorescence was detected;
This indicated that a substantial amount of the fluorescein-labeled antibody reached the registration layer, whereas the test sample showed significantly less fluorescence, indicating that the fluorescein-labeled antibody was immobilized by the antigen and did not reach the registration layer. It was shown that In contrast to the kinetic techniques presented in the prior art mentioned above, the present invention uses endpoint techniques. As used herein, the term "endpoint technology" is used to refer to a depiction based on a chemical reaction that produces a detectable product whose total amount is substantially fixed after some specified time in which the reactants are substantially depleted. is intended. Endpoint techniques operate with ambient levels of analyte and stoichiometric amounts of reagents, and are inherently less sensitive to temperature fluctuations than kinetic techniques. For example, the displacement release mechanisms described in US Pat. Nos. 3,992,158, 4,422,335 and 4,144,306 are essentially kinetic assay systems. This is because a large excess of reagent is required to drive the reaction to a measurable rate. Kinetic techniques are temperature and time sensitive and require complex instrumentation to determine rate constants. The novel methods and elements of the present invention provide inherently faster reaction times. This is because the association reaction rate is many orders of magnitude faster than dissociation, which is a necessary prerequisite for the displacement release used in the patent.