JPS6348312B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the field of analytical test elements and test methods, such as those useful in manual and automated diagnostic systems, and more particularly to the field of analytical test elements and test methods, such as those useful in manual and automated diagnostic systems, and more particularly to the field of analytical test elements and test methods, such as those useful in manual and automated diagnostic systems, and more particularly, to the field of analytical test elements and test methods that are useful in manual and automated diagnostic systems, and more particularly, to the field of analytical test elements and test methods, such as those useful in manual and automated diagnostic systems. The present invention relates to a multilayer analytical element useful for qualitative and quantitative analysis.

Test devices in the form of test strips and similar solid state analytical elements have become commonplace in the analysis of various types of samples, particularly biological fluids. Test strips constructed to detect clinically important substances in biological fluids such as serum and urine have become useful in the diagnosis of disease.

The various test strips range from the most familiar PH test strips to various urinary and blood components such as glucose, protein, occult blood, etc. (as described in U.S. Pat. It has been known and used for many years in a wide range of fields, including in vitro diagnostic testing devices. Although the reagent compositions found in such test strips often have limited sensitivity, they interact with the component or components to be measured by direct chemical reactions; It has been applied to the detection of substances present in liquid samples at concentrations in the millimolar range or higher.

(a) A basic multilayer monolithic analytical element is described in US Pat. No. 3,092,465. Such multilayer elements use, in particular, an absorbent fibrous carrier impregnated with one or more reagents, including a color former, coated with a semipermeable membrane. . Upon contact with the test liquid, the analyte passes through the membrane to the fibrous carrier, resulting in an amount of color that is related to the concentration of the analyte. This membrane prevents the passage and absorption of certain interfering components, such as red blood cells, that could interfere with the accurate reading of the color resulting from the test.

Other multilayer integral analytical elements are described in U.S. Pat.
Described in No. 3992158. Such an element is
receiving a liquid sample and spreading the sample within the spreading layer of the element, resulting in an analyte, other appropriate sample components or products of the analyte within the element; and In the presence of an object,
Techniques such as spectrophotometry, fluorescence, etc. are used to produce analytical results that can be measured quantitatively by automated equipment. Such devices include a spreading layer and a reagent layer containing a reactive, otherwise interactive substance whose activity promotes a photometrically detectable change within the device, such as a color change. It can be composed of.

U.S. Pat. No. 4,042,335 discloses the following: (1) a reagent layer that reacts with the analyte to form diffusible, detectable species; (2) a layer that is transparent and opaque to the detectable species; and (3) a non-fibrous light-transmitting display layer in which a detectable species is detected. This element is thus read below.

U.S. Pat. No. 4,066,403 (Re30267) discloses (1) a reagent that reacts with an analyte to produce a degradation product; and (2) a reagent that reacts with a degradation product or intermediate to produce a detectable change. and as an improvement thereof, a barrier composition separating reagent (1) from reagent (2), substantially homogeneously permeable to degradation products and substantially impermeable to interfering substances. It relates to elements that are opaque to objects. So what this does is add a "super" layer between the "reagent" layer and the "display" layer.

U.S. Pat. No. 4,144,306 describes a device in which the reagent layer comprises a non-diffusible material containing a pre-formed detectable moiety that becomes liberated and diffusible in the presence of an analyte. Regarding. The display layer receives the diffusible species. The layers within the device are configured such that preformed detectable portions liberated from the reagent layer can be selectively detected within the device.

U.S. Pat. No. 4,166,093 describes (1) a radiation-transparent reagent layer that reacts with the analyte to produce a detectable species; and (2) a porous radiation-transparent layer that is transparent to the analyte. The present invention relates to an element having a line shielding layer. As an improvement thereof, it further comprises (3) a layer between the reagent layer and the porous radiation shielding layer that is radiation transparent and prevents the migration of detectable species. This anti-migration layer is transparent to the analyte, but prevents the migration of detectable species to the radiation shielding layer.

(b) Efforts have been made to overcome the inconvenience and drawback of solid phase test devices being applied to heterogeneous binding analysis methods and requiring a separation step. This type of commonly used solid-phase test device consists of a nonporous surface, such as the interior surface of a test tube or other container, to which the antibody is immobilized or bound by adsorption or covalent bonding. It is painted. US Patent No. 3826619; 4001583
Nos. 4017597 and 4105410 relate to the use of antibody-coated test tubes in radioimmunoassays. The solid phase test device is also
It has been used in heterogeneous enzyme immunoassays (US Pat. Nos. 4,016,043 and 4,147,752) and heterogeneous fluorescence immunoassays (US Pat. Nos. 4,025,310 and 4,056,724; and British Patent No. 1,552,374).
The use of such test devices for heterogeneous specific binding analysis is described in US Pat. No. 4,135,884 for the so-called "gamma stick".
This method is exemplified by the method in No. This test device contains an antibody reagent and is brought into contact with the liquid sample and the residual reagents of the reaction system, essentially the labeled complex. After a certain standing time, the solid phase test device is physically removed from the reaction solution and a label is placed either in the solution or on the test device.
is measured.

Similar test devices in which the antibody reagent is held in a matrix such as a gel or paper web are described in U.S. Pat. Nos. 3,925,017; 3,970,429;
No. 3966897; No. 3981981 and No. 3888629, and West German Published Application No. 2241646. Similarly, test devices for heterogeneous specific binding analysis are known in which antibodies are immobilized on a matrix held in a permeable column (U.S. Pat. Nos. 4,036,947; 4,039,652; 4,059,684). issue;
No. 4153675; and No. 4166102). This test tool includes
Typically, not all of the necessary reagents to carry out the analysis are included; it is merely a means to facilitate the necessary separation steps.

Finally, a test device for heterogeneous specific binding analysis is disclosed, in which most or all of the necessary reagents are contained in the same carrier element, and the contact between the reagents and the sample and between the free and bound phases. separation is done by movement in capillary tubes along the carrier element (US Pat. Nos. 3,641,235; 4,094,647 and 4,168,146).
The test devices described in these patents are generally difficult to manufacture and difficult to reproduce due to the complex nature of the many chemical and physical interactions that occur along the carrier element during the course of the analysis. It is thought that it is difficult to obtain sex. Additionally, other attempts at heterogeneous immunoassays are illustrated in US Application No. 973,669, published as European Patent Application No. 0013156.

The application of homogeneous specific binding analytical reagent systems to solid-state test devices would be of great benefit to those who routinely use such analytical systems. Measurements of ligands present in very low concentrations in liquid samples can be obtained either by contacting a test device with the sample and observing it with the naked eye, or by instrumental means. The process of measuring the signal will be simplified. The reagents are supplied in a solid state and do not require storage, dispensing or mixing of liquid reagents as is required when using prior art test kits. A solid state test device would also have much greater compatibility with automation than prior art liquid systems.

GB 1552607 describes homogeneous specific binding using a variety of new labels, including chemiluminescent labels, enzyme substrate labels and coenzyme labels, as generally cited herein. The analysis system is disclosed. On page 23, lines 12 et seq. of this patent, it is stated that analytical reagents can be stored in liquid-containing containers or in insoluble, porous, and preferably absorbent matrices, fleece or lint; gel materials; etc. It has been suggested that they be included in various carriers. This lacks detailed teachings on how to apply homogeneous specific binding assay reagent systems to solid state test devices.

DE 2537275 describes a homogeneous specific binding assay reagent system and mentions the possibility of using strips or slides containing antibodies in carrying out the assay. In this suggestion, the labeled complex is first mixed with the sample, and then the test device containing the antibody is contacted with the reaction mixture. After a suitable incubation period, it is proposed to wash the test device with a buffer, dry it and then measure the signal (fluorescence). Thus, this West German Published Application describes the already well-known heterogeneous specific binding analysis technique in which the test device is immersed in a liquid reaction mixture, incubated, taken out, washed, and finally read. The test equipment and analysis methods are presented. Furthermore, the test device proposed herein does not include all of the binding assay reagents in the carrier element. In particular, it is proposed to include only the antibody in the carrier, and the labeled complex is added to the sample to be tested prior to contact with the proposed test device.

U.S. Pat. No. 255,521, filed on April 20, 1981 and pending, discloses the presence or ability of a ligand to bind (hereinafter referred to as "ligand binding capacity") in a liquid test sample. ), the method comprises: (1) adding to a liquid sample a ligand complex comprising a ligand or a binding analog thereof chemically coupled to a label; (2) producing a homogeneous specific binding assay system that, when combined with a labeled complex, produces a detectable response that is a function of the presence of the ligand or ligand binding capacity, thereby producing a response; It is characterized by comprising the steps of (3) contacting a test device consisting of a carrier matrix containing a reagent that produces a reaction, and (3) measuring the response.

No. 202,378, filed and pending on October 30, 1980, describes a homogeneous specific binding test device, method for its preparation, and determination of ligands or ligand binding capacity in liquid samples. Discloses how to use it to. This includes, for example, (a) reagents for homogeneous specific binding test systems that produce a detectable response that is a function of the presence of the ligand or ligand binding capacity in the sample; and (b) reagents. The present invention includes a test device for measuring a ligand or a ligand binding ability in a liquid sample consisting of a solid carrier member and a solid carrier member.

U.S. Pat. ii) a reagent composition comprising a complex with a specific binding pair for the ligand, where the label interacts with a detectable response or with a detection system to produce a detectable response - that is, the label complex binds (b) a carrier containing said complex; Discloses a homogeneous specific binding test device for use in measuring.

Problems present in prior art devices have been recognized and avoided or overcome by the multilayer analytical device of the present invention. Since the response of the device is read from the device surface remote from the reagent layer, i.e. from the support layer surface, the shielding layer used in prior art devices is free from the ligands, the reagents of the reagent layer or the products of their interaction. It is required to be transparent to

This problem arises because electromagnetic radiation, such as that emitted in reflective and fluorescent systems, must pass through a support layer such as a polystyrene or polyester layer (in prior art devices, the radiation must pass through the support layer). ). Some of the electromagnetic radiation, such as light, that passes through the support layer is trapped within the layer. As such, it essentially behaves as an optical fiber. Thus, the sensed amount of electromagnetic radiation, such as light, does not accurately indicate the amount obtained from reactions occurring within the device. Response dose results etc. therefore do not fully indicate the amount of electromagnetic radiation from the response to the ligand within the reagent layer. In the case of devices that are read using fluorescent systems, a certain amount of the emitted light is trapped, so this problem becomes more pronounced in the reliability of the results obtained when the ligand concentration is low. have a significant impact. For devices read using reflective systems, this problem is even more severe when the ligand concentration is high.

These problems are avoided or overcome in the multilayer analytical element of the present invention. In overcoming these problems, the devices of the present invention emit enhanced electromagnetic responses or signals compared to prior devices. Even more remarkable is that the ratio of the signal radiation S to the emitted background radiation B is increased such that there is no interfering background radiation, and the ratio is as shown in the example below. , will be at least 10 times the order of magnitude.

Thus, according to the present invention, a detectable response is generated in response to a ligand or a ligand-binding capacity in a sample in order to detect a ligand or a ligand-binding capacity in a liquid sample. It is a multilayer analytical element of the type that has a reagent layer containing the reagent indicated above, a radiation diffusion/shielding layer, and a support layer, and its improvement is that the radiation diffusion/shielding layer has (a) a reagent layer and a support layer. (b) impermeable to the ligand, the reagents of the reagent layer, and the products of their interaction; and (c)
A multilayer analytical element is provided in that it is inert to the ligand, the reagents of the reagent layer, and the products of their interaction.

For the sake of clarity, specific terminology is used in the following description, which refers only to particular embodiments of the invention selected for illustration.
It is not intended to limit the scope of the invention.

1. Ligand The term ligand refers to components of body fluids and drugs or other substances present in such body fluids. A number of such possible ligands are illustrated below.

Reagent compositions include blood such as ascorbic acid, liver juice acids, bilirubin, cholesterol, creatinine, glucose, lactic acid, phospholipids, triglycerides, urea nitrogen (BUN) and uric acid;
Known for plasma or serum ligands. Additionally, amylase, cholinesterase, creatine phosphokinase (CPK), dehydrogenases (dehydrates hydroxybutyrate, isocitrate, lactic acid, and malate), lipase, phenylalanine, transaminases (dehydrates glutamate to oxaloacetate, and converts glutamate to oxaloacetate). pyruvate), acid and alkaline phosphatases, gamma-glutamyl transpeptidase, leucine aminopeptidase and erythrocytic enzymes (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and Measurement of enzyme ligands in blood chemistry such as pyruvate kinase) is also important. Blood protein ligands such as albumin, cryoglobins, components of the coagulation and fibrinolytic systems, complement factors, and cellular and serum immune agents such as interferons and immunoglobins have also been tested. is possible, and is discussed in detail below in connection with homogeneous specific binding assays.

Similarly, reagent compositions for measurements based on urine chemistry are known. In the field of urinary chemistry, such ligands generally include ascorbic acid, albumin, creatine, creatinine, glucose, bile acids, pyrrubin, proteins, ketones, occult blood, nitrite, amylase, and phenylpyruvate. .

The analytical element of the present invention furthermore detects all the ligands for which specific binding partners are present and, conversely, the ability of the liquid medium to bind to the ligands (generally, the ability of the liquid medium to bind to the ligands). (due to the existence of a binding partner for). This ligand is typically used for peptides, polypeptides,
It may be a protein, carbohydrate, glycoprotein, steroid, or other organic molecule for which a specific binding partner exists in a biochemical system or can be synthesized. In functional terms, these ligands are usually used for antigens and antibodies against them; haptens and antibodies against them; and hormones, vitamins, metabolites and drugs, and their Selected from the group consisting of receptors and binding substances. Typically, the ligand is an immunologically active polypeptide or protein with a molecular weight of 1,000 to 1,000,000, such as an antibody or an antigenic polypeptide, or a molecular weight of 100 to 1,000,000.
There are 1500 haptens.

Representative polypeptide ligands are angiotensin I and C-peptide, oxytocin, vasopressin, nyurophysin, gastrin, secretin, bradykinin, and glucagon.

Typical protein ligands include protamines,
Includes mucoproteins, glycoproteins, globulins, albumins, scleroproteins, phosphoproteins, histones, lipoproteins, chromoproteins, and nucleoproteins. Examples of specific proteins include prealbumin, α ₁ -lipoprotein, human serum albumin, α ₁ -glycoprotein, transcortin, thyroxine-binding globulin, haptoglobin, hemoglobin, myglobin, celluloplasmin, α ₂ - Lipoprotein, _α2 -macroglobulin, β-lipoprotein, erythropoietin, transferrin,
homopexin, fibrinogen, IgG,
IgM, IgA, IgD and IgE and fragments thereof, such as immunoglobulins such as Fc and Fab, complements, clotting factors such as prolactin, fibrinogen, thrombin, etc., insulin, melanotropin, somatotropin, thyrotropin , follicle stipulated hormone, leutinizing hormone, gonadotropin, thyroid stimulating hormone, placental lactogen, intrinsic factor, transcobalamin, alkaline phosphatases, cholinesterases, glutamate-oxaloacetate transaminase, glutamate-pyruvate transaminase and uropepsin. serum enzymes such as endorphins, enkephalins, protamines, tissue antigens, bacterial antigens, as well as hepatitis-related antigens (e.g. HBsAg,
viral antigens such as HBcAg and HBeAg).

Typical hapten ligands include common drugs, metabolites, hormones, vitamins and similar organic compounds. Hapten hormones include thyroxine and triiodothyronine. Vitamins include vitamin A,
B, such as B ₁₂ , C, D, E and K, folic acid and thiamine. Drugs include, for example, gentamicin, tobramycin, amikacin,
Aminoglycosides such as sisomicin, kanamycin and netilmicin, penicillin,
Antibiotics such as tetracycline, terramycin, chloromycetin; and actinomycetin; nucleosides and adenosine diphosphate (ADP), adenosine triphosphate (ATP), flavin mononucleotide (FMN), nicotinamide adenine Nucleotides such as dinucleotides (NAD) and their phosphate derivatives (NADP), thymidine, guanosine and adenosine, prostaglandins; estrogens, sterogens, androgens such as estriol and estradiol , digoxin, digitoxin, and steroids such as adrenal stimulant steroids;
Phenobarbital, phenytoin, primidone, etosuximide, carbamazepine, valproate, theofylline, caffein, propranolol, procainamide, quinidine, amitriptyline, cortisol, desipramine, disopramide, doxepin, doxorubicin, nortriptyline, methotrexate, isopramine, lidocaine, procainamide cain Others such as amide, N-acetylprocainamide, amphetamines, catecholamines and antihistamines are included.

The liquid medium to be tested may be a naturally occurring or artificially formed liquid believed to contain the ligand, usually a biochemical liquid or a dilution thereof. Biochemical fluids that can be tested include serum, plasma, urine, saliva, and amniotic and cerebrospinal fluid.

2 Radiation Scattering/Shielding Layer The position of the radiation scattering/shielding layer in relation to the reagent layer and the support layer is important in the present invention. The properties of the radiation scattering and shielding layer depend on the components with which it is prepared, and these properties are likewise important to the present invention.

The radiation shielding layer of the present invention is a radiation scattering/shielding layer interposed between the reagent layer and the support layer. It can be brought into direct contact with one side of the reagent layer; the signal emitted from the reagent layer is then read from the other side of the reagent layer. In this way, the radiation signal does not need to pass through any other layers or materials for detection besides the reagent layer from which it is emitted. If the reagent layer does not bond well to the radiation scattering/shielding layer, the radiation scattering/shielding layer may be treated with a suitable treatment, such as treatment with sodium hydroxide, to improve the radiation scattering/shielding layer. It is sometimes possible to treat or activate the surface to improve the bond between the reagent layer and the radiation scattering and shielding layer. Alternatively, a subbing layer can be placed between the radiation scattering and shielding layer and the reagent layer to improve the bonding or adhesion of the reagent layer.

The characteristics or properties of the radiation diffusion/shielding layer are:
It is opaque, relatively thin and impermeable to and inert to the ligand, the reagents of the reagent layer and the products of their interactions. The radiation diffusion and shielding layer preferably contains a white or light colored pigment homogeneously dispersed throughout the layer or at least on the surface of the layer adjacent to the reagent layer or subbing layer. More particularly, the radiation diffusion/shielding layer is a radiation-opaque material contained in a suitable resin or polymeric material compatible with the substrate or support material on the one hand and the reagent layer or subbing layer on the other hand. It can be composed of pigments. If polymeric in nature, the radiation diffusion/shielding layer is a homopolymer or copolymer.

The radiation scattering/shielding layer can be of any suitable thickness, but typically will be about 0.0002 to about 0.02 inch thick.

Examples of radiopaque pigments that can be used include titanium dioxide, barium sulfate, zinc oxide, magnesium oxide, zirconium dioxide and the like. Typically, the upper limit for the amount of pigment present will be less than 40% solids content, and the lower limit will be greater than 2% solids content.

Examples of suitable resins that can be used in the radiation diffusion/shielding layer include methyl vinyl ether/maleic anhydride copolymer; ethylene/maleic anhydride copolymer; octadecenyl-1.
Examples include anhydride resins including maleic anhydride copolymer; octadecyl vinyl ether/maleic anhydride copolymer; styrene/maleic anhydride copolymer.

Examples of other polymeric materials that can be used in the radiation diffusion/shielding layer include polyvinyl acetate, polyvinyl methacrylate, polybutadiene, polychloroprene, polyvinylpyrrolidone, and the like.

3. Homogeneous specific binding test Reagents for all homogeneous specific binding test systems are:
It can be included in the test device of the present invention. In general, techniques for homogeneous specific binding assays are based on the specific interaction between (1) a complex of a binding component and a label, and (2) a binding partner for the binding component in the complex. , is based on special interactions such that when a labeling complex is bound by a binding partner, the labeling properties are matched compared to when the complex is not so bound. of the sign,
The affected property may be any measurable property, such as a chemical or physical property of the label. In some cases, the affected property is chemical reactivity in a predetermined reaction, including the formation or breaking of chemical bonds, covalent or non-covalent bonds. In other cases, the property affected is a physical property of the label that can be measured without chemical reaction.

In many cases, the test device of the invention includes:
Homogeneous specific binding test reagents that interact in an immunochemical manner with the ligand or its binding capacity in the sample will be included. That is, in the sample,
There may be an antigen-antibody or adherent-antibody relationship between the reagents and/or the ligand in the sample or its binding capacity. Such tests are therefore named immunoassays, and the specific interaction between the labeled complex and its binding partner is immunochemical binding. Thus, in such instances, the binding component of the labeling complex is an antigen, hapten or antibody (or fragment thereof), and the binding partner is the corresponding immunochemical binding partner. However, traditionally, other binding interactions between the labeled complex and the binding partner have been associated with hormones, vitamins,
It is well known to serve as the basis for homogeneous specific binding studies involving binding interactions between metabolites and drugs and their respective receptors and binding substances.

When a sample is being tested to determine the presence or amount of a particular ligand therein, reagents for homogeneous specific binding testing techniques typically include (1) chemically bound to a label; a label complex consisting of a ligand or a binding analog thereof; (2) a binding partner for the ligand, such as an antibody or fragment thereof, a natural receptor protein; and (3) a label within the label complex. It consists of all the auxiliary reagents needed to measure the substance. A limited amount of binding material is introduced so that all the ligands in the sample will compete with the labeled complex for binding to the binding partner. The distribution of the label between bound and free species will therefore determine the degree of detectable response from the label, which in turn will be a function of the presence of the ligand. Another method for measuring ligands is when the labeled complex consists of a labeled binding partner of the ligand, such that upon binding to the ligand the label is affected according to its detectable response. is given to. When testing the ligand binding capacity of a sample, the label complex consists of a ligand or a binding analog thereof chemically bound to the label, thereby making it possible to bind the label complex. The capacity of the sample to be detected measures the effect on the detectable signal from the label, eg, due to the presence of a binding partner of the ligand in the sample.

A number of different homogeneous specific binding test systems are known in the art, and the following, without limiting the scope of the invention, describes some of them for use with the test device of the invention. This is an example of such a system. The following systems are designated by the nature of the label used.

(a) Enzyme prosthetic group label In this system, the label is a prosthetic group of the enzyme, and the label is a catalytically inactive apoenzyme that, together with the prosthetic group label, produces an active enzyme (holoenzyme). Capacity is influenced by the binding of the label complex to its binding partner. The activity of the holoenzyme obtained is measured by a conventional detection system, ultimately yielding a detectable signal. A test system of this type is described in pending US Application No. 45423 (corresponding to GB 2023607) filed June 4, 1979. Particularly preferred prosthetic group labeled test methods include:
Flavin adenine dinucleotide (FAD) is used as a label and apoglucose oxidase is used as an apoenzyme. The activity of the resulting glucose oxidase can be measured by a colorimetric detection system consisting of glucose, peroxidase, and an indicator system that produces a color change in response to hydrogen peroxide.

In this preferred test method, the FAD-labeled complex preferably has the formula: (In the formula, Riboflavin (-Phos-) ₂ Ribose is FAD
represents a riboflavin-pyrophosphate-ribose residue in which R is a linking group and L is a linking moiety, such as a ligand or an analog thereof. ). An example of such a complex is the 1980
The FAD-theophylline complex disclosed in pending US Application No. 202378, filed on October 30th.

(b) Enzyme Substrate Label In this system, the label is a substrate for the enzyme, and the ability of the enzyme to act on this substrate-label complex is determined by the binding of the label complex to its binding partner. By doing so, it is selected to be influenced in either a positive or negative sense. The action of the enzyme on the substrate-label complex depends on several aspects (properties), such as chemical inertness in the indicator reaction or photometric properties such as fluorescence or light absorption (color). produce a product that is distinguishable in its usual physical or physical properties. This type of test system is described in pending U.S. Application No. 1, filed April 10, 1978.
894836 (corresponding to West German Publication No. 2618511) and U.S. Application No. 87819 filed October 23, 1979; and Anal.Chem.48:1933
(1976), Anal.Biochem.77:55 (1977) and
Clin.Chem.23:1402 (1977).

A particularly preferred substrate-label test method has the structural formula: (wherein R is a binding group and L is a binding moiety such as a ligand or an analogue thereof), thereby:
The ability of the enzyme β-galactosidase to cleave a conjugate to form a product distinguishable by its fluorescence indicates that the complex is Prevented by binding to a binding partner.

(c) Coenzyme labels In the label complex in this system, the label part consists of a functional part having coenzyme activity, and the ability of such a coenzyme label to participate in the enzymatic reaction is determined by the binding of the label. Affected by the bond with the other party. The rate of the enzymatic reaction that occurs can be measured by conventional detection systems that result in a detectable signal. This type of test method was introduced in April 1978.
No. 894,836, filed and pending on May 10; and Anal.Biochem.72:
271 (1976), Anal.Biochem.72:283 (1976)
and Anal.Biochem.76:95 (1976).

(d) Labels that affect enzyme activity The label complex in this system consists of a functional moiety that affects enzyme activity, such as an enzyme inhibitor or promoter, and a label that affects enzyme activity. The ability of the labeling complex to affect the enzymatic activity is influenced by the binding of the labeled complex to its binding partner. The rate of this enzymatic reaction can be measured by conventional detection systems that ultimately produce a detectable signal. This type of test method is described in US Pat. No. 4,134,792.

(e) Enzyme label group In this system, the label is an enzyme and the activity of the enzyme label is influenced by the binding of the label complex to its binding partner. The enzymatic activity obtained can be measured by conventional detection systems that ultimately produce a detectable signal. Test methods of this type are described in US Pat. Nos. 3,817,837 and 4,043,872.

(f) Extinguishable Fluorescent Labels The labeling complex in this system consists of a fluorophore in its labeling portion, and the fluorescence is generated by the binding partner of the labeling complex, which is generally a protein such as an antibody. When combined, they disappear to some measurable extent. This fluorescent label is directly measured with its fluorescence being a detectable signal. This type of test system is described in US Pat. No. 4,160,016 and J.Clin.Path.

(g) Fluorescent polarizing labels The label in this system is also a fluorophore; however, the properties that are affected are the binding of the label complex to its binding partner, which is usually a protein such as an antibody. This is the perspective of fluorescence based on This type of test system is J.Exp.
Med.122:1029 (1965).

(h) Chemically Excited Fluorescent Labels In this system, the label is again a fluorophore, but a fluorophore that is to be chemically excited to an energy level at which it fluoresces. The ability of body labeling is influenced by the binding of the labeling complex to its binding partner. Chemical excitation of the label is generally accomplished by exposing the fluorophore label to high energy compounds that are formed in situ. This type of test system is
No. 4580, filed and pending on January 18, 1979.

(i) Dual-antibody sterically hindered labels Other analytical systems are described in U.S. Pat. No. 3,935,074 and
This is a double antibody immunoassay system described in No. 3998943. The labeling complex consists of two epitopes, one of which participates in the immunological reaction with the ligand and anti-ligand antibody, the other capable of binding by a second antibody, provided that , the two antibodies cannot bind to the label complex at the same time. The second epitope is a fluorophore, and either its fluorescence is quenched upon binding of the second antibody, or it is conjugated with a label of the second epitope for binding to the second antibody. It may also participate in competitive binding reactions.
A variety of sensing systems are possible, such as those described in the aforementioned patents. Related test systems are described in US Pat. Nos. 4,130,462 and 4,161,515 and British Patent No. 1,560,852.

(j) Energy Transfer Labels In this system, the label is one of a pair of donor and acceptor that participates in energy transfer, and its binding partner binds to the other of the pair. do. Thus,
When a labeled complex binds to a binding partner, the energy signature of the donor moiety of the pair changes due to transfer to the acceptor moiety. Typically, the donor is a fluorophore and the acceptor is its quencher, which may or may not be a fluorophore as well. In such embodiments, the detectable signal is fluorescence, although other detection systems are also possible. Such test systems are described in US Pat. No. 3,996,345;
4174384; and 4199559, and UK Patent No. 2018424.

(k) Other Labels Other previously described homogeneous specific binding test systems that can be used in the present invention include (i) non-enzymatic catalysts such as electron transfer agents (see U.S. Pat. No. 4,160,645; ); (ii) non-enzymatic chemiluminescent agents (mentioned above,
See pending U.S. Application No. 894,836. ); (iii) "chnnneling" markings (see GB 2019562); (iv) "partile" markings (see GB 2019562); and (v) markings. Labels such as liposome particles (see US Pat. No. 4,193,983) are used.

4 Reagent Layer Also provided herein is a method of providing a reagent layer on the radiation diffusing/shielding layer or, if a subbing layer is used, on the subbing layer. The test device of the present invention may be manufactured by any suitable method such as printing or spraying the reagent composition onto the radiation scattering/shielding layer or subbing layer, or by using any of the known film forming techniques. Can be done.

If the reagent layer actually consists of multiple layers,
Such layers can be maintained in laminar relationship with an adhesive that allows fluid passage between the layers, if desired. However, it is usually not necessary to use adhesives to adhere one layer to another. In the manufacture of integral analytical elements using film forming equipment, the layers can be preformed separately and laminated to form the overall element. The material of the film layer may be a composition comprising a plasticizer and a polymer suitable for imparting dimensional stability. Layers produced in this manner are typically applied from a solution or suspension onto a surface, from which the dried layer can be physically peeled off. However, a convenient method that can avoid the problems of multiple peeling and lamination processes is to apply the first layer on the surface to be peeled or, if necessary, on the support layer, and then to apply the first layer on top of those already applied. It is the application of layers directly one after the other. Such application can be accomplished by hand, with a blade applicator, or by machine, using techniques such as dipping or beading. If mechanical coating methods are used, it is often possible to coat adjacent layers simultaneously using hopper coating methods, which are well known in the manufacture of light-sensitive photographic films and paper.

A blush polymer layer can be used as the film layer material. This film is formed on the substrate by dissolving the polymer in a mixture of two liquids. One of the liquids has a low boiling point and is a good solvent for the polymer, and the other has a high boiling point and is a non-solvent or at least a poor solvent for the polymer. Such a polymer solution is then applied onto a substrate and dried under controlled conditions. Low-boiling solvents evaporate more easily and the coating is enriched in liquids that are poor solvents or non-solvents. As evaporation progresses, under certain conditions the polymer becomes a porous layer. Many types of polymers can be used alone or in combination for the preparation of porous fogged polymer layers for use in the present invention. Typical examples include polycarbonates, polyamides,
Included are polyurethanes and cellulose esters such as cellulose acetate. To form such a layer containing the labeled complex or other reagent, a coating solution or suspension containing the matrix and the encapsulated active agent is prepared;
It can be applied as discussed herein and dried to form a dimensionally stable layer.

The thickness of the reagent layer and its degree of permeability can vary widely depending on the actual application. Dry thicknesses of about 5 microns to about 100 microns have been convenient, although more widely varying thicknesses may be preferred under certain circumstances. For example, if relatively large amounts of interactive substances, such as polymeric substances such as enzymes, are required, it may be preferable to prepare slightly thicker layers.

1 such as anionic and nonionic surfactants
It is advantageous to include one or more surfactants in the reagent layer. For example, they are
It improves the coatability of layer-forming formulations and increases the magnitude and extent of wetting in layers that are not easily wetted by liquid samples without auxiliary agents such as surfactants. Additionally, in a chosen assay, it may be desirable to include materials that can be made inert, by chemical reaction or otherwise, that are potentially harmful to the assay.

Below, some preferred approaches to the preparation of reagent layers are described.

(a) Multilayer trial This trial concerns a method for forming a reagent layer of a homogeneous specific binding analysis element for measuring a ligand or ligand binding ability in a liquid sample. This method involves the inclusion of a composition comprising a labeled complex consisting of a labeled component bound to a ligand moiety (molecule) or its specific binding analogue, and a reagent that is mutually reactive with the labeled complex. It is carried out with This reagent is
For example, it consists of a specific binding partner for the ligand and a moiety that interacts with the label complex to cleave the label moiety from the ligand moiety or its specific binding analog.

The reagents can be used in the form of their respective solutions, which are capable of substantial interaction during the preparation of the test device and thus do not react prematurely. In a preferred embodiment, a first reagent is incorporated into the layer using an aqueous immersion;
For other reagents, suitable organic solvents such as toluene, acetone, chloroform, methylene chloride, n-propanol and ethylene dichloride are used. This layer is fixed by evaporating the organic solvent.

An example of this preferred embodiment is the preparation of a complex of β-galactosyl umbelliferone in a liquid sample by applying an antiserum against the ligand or ligand analogue complex, β-galactosidase and the ligand. A method for preparing a homogeneous specific binding test device for determining the binding capacity of a β-galactosidase or a ligand, the method comprising: (a) applying an antiserum against β-galactosidase and the ligand in an aqueous liquid; , and (b) then applying the β-galactosyl umbelliferone-ligand or ligand analog complex in acetone.

(b) Multi-region reagent layer The multi-region reagent layer includes (a) some, but not all, of the reagents of the specific binding analytical system to be used, which are included in the first or upper region; and (b) by incorporating the remaining reagents into the second or underlying region, (c) fixing the individual regions, e.g. by drying, and (d) fixing them in a laminar manner in a laminated relationship with each other. It is prepared by

Both the first layer and the second layer have a pair of opposing surfaces. One side of the first layer is one side of the second layer
and the sample is applied to the other side of either of the layers. A stacked relationship means that fluids, whether liquids or gases, can pass between the overlapping surfaces of these layers. Such layers may be adjacent to each other or separated by intervening layers. Any intervening layers must not obstruct passage between all layers.

(c) Freeze-drying method This method consists of incorporating reagents that cannot coexist in one layer of analytical elements and preventing reactions between the reagents. For example, a first group of reagents is applied by freeze-drying or at elevated temperatures to provide a treatment layer. A second group of reagents, including those that react with the first group under ambient atmosphere, is applied and the device is rapidly frozen. Freezing prevents premature reaction and subsequent removal of water by freeze drying prevents premature reaction when this layer is brought back to room temperature.

In a preferred embodiment, a group of reagents may be added to the layer in the form of an aqueous solution and dried. A second group of reagents is added in the form of an aqueous solution, which is then rapidly frozen and freeze-dried to remove the water. This method is applied when several reagents that are only soluble in water are included and mutual reactions between these reagents are prevented. Furthermore, no organic solvents are used in this method. This is because certain organic solvents can react undesirably with some reagents (eg, enzymes).

This method allows for the formation of devices using reagents for homogeneous specific binding studies where all reagents are provided within a single layer device.

(d) Preformed Complex Method Competition between a labeled ligand for binding to a sample ligand and a binding partner (here exemplified by antibody “Ab”) is determined by the formula: Ligand Ab: It can be summarized as: Ligand + + Label-Ligand Ab: Label-Ligand + Ab.

In the system shown above, the antibody and label complex are kept separate until the introduction of the sample. This aspect of the described invention is directed to the reverse reaction with the ligand, as shown by the following formula: Ligand Ab:Ligand Re-equilibration is used, where the displaced label complex is related to the sample ligand concentration. The advantage of this is that all reagent components are combined in one contained medium, providing a system that only requires the addition of the sample to be tested.

Thus, this method provides a method for preparing a reagent layer of a homogeneous specific binding test device for measuring a ligand in a liquid sample; forming a complex between a complex consisting of a labeling moiety bound to a ligand or a specific binding analog thereof and a specific binding partner for the ligand; and
(b) consists of applying the complex.
In this method, the formation of the complex consists of bringing together a labeled complex and a specific binding partner for it and allowing the complex, binding partner and complex to reach a certain equilibrium state.

More specifically, this layer is prepared by incubating a given complex with its respective antiserum for a short period of time, for example 15 minutes. Other reagents are then added and incubated for an additional period of time. The solution so formed is then allowed to stand.

5 Support Layer As already mentioned here, the integral analytical element also includes a support. The support may be opaque, translucent or transparent to light or other energy. The support chosen for a particular element will be chosen regardless of the intended mode of signal detection. Preferred supports include those made of polystyrene or similar plastic materials.

6. Preparation of Multilayer Devices The present invention further provides a reagent layer that includes a reagent responsive to a ligand in a sample or its ligand binding ability and provides a detectable response, a radiation diffusion/shielding layer, and a reagent layer that provides a detectable response. Provided is a method for preparing a multilayer analytical element for detecting a ligand or ligand binding ability in a liquid sample, the type having a support layer and each layer having opposing surfaces, the method comprising: (1) impermeable to (a) the ligand, the reagents of the reagent layer, and the products of their interaction; and (b) the ligand;
The surface of the support layer is fixed to the surface of the radiation diffusion/shielding layer which is inert to the reagents in the reagent layer and the products of their mutual reactions, and (2) the radiation diffusion/shielding layer is inert. It consists of the step of fixing the surface of the reagent layer to the opposing (opposite) surface.

In one embodiment, affixing a surface of the support layer to one surface of the radiation diffusing and shielding layer comprises:
forming a radiation diffusing and shielding layer on that side of the support layer. In other embodiments, bonding one side of the support layer to one side of the radiation diffusing and shielding layer forms the radiation diffusing and shielding layer, and then attaching the thus formed radiation diffusing and shielding layer to the side of the support layer. It consists of fixing a radiation diffusion/shielding layer.

7. Detectable Responses As previously noted, many of the recently developed homogeneous specific binding test systems detect color changes, chemiluminescence, or It provides or can be easily adapted to provide a detectable response such as fluorescence.

The term "detectable species" and similar terms as used herein means that an atom, a chemical group (i.e., part of a molecule), or itself is
represents a compound that is indirectly detectable, and
As used herein, the term "detectable response" and similar terms refer to a detectable manifestation of the presence of such species. For example,
Fluorescence, phosphorescence, chemiluminescence, changes in light absorption resulting in a color change in the visible range, or reflection in the visible spectrum, changes associated with light absorption or reflection outside the visible range, such as in the ultraviolet or infrared range, etc. Examples include electromagnetic wave signals. As will be apparent to those skilled in the art, the term "detectable response" as used herein is intended in its broadest sense. In addition to electromagnetic signals,
The term "detectable response" means any observable change in a parameter of a system, such as a change or appearance of a reactant, an observable precipitation of a component in a test system, or Changes in any other parameters can also be mentioned, even in the test sample being a reagent system. Such other detectable responses also include electrochemical responses and calorimetric responses. Additionally, this detectable response can be observed directly through the senses or by auxiliary sensing such as spectrophotometers, ultraviolet sensing devices, fluorometers, fluorometers, and other similar sensing systems. It is something that can be observed using a certain method. Preferably,
Such detectability conveniently imparts the entire amount of detectable species without affecting the amount of diffusible products resulting from analyte interactions that are the basis of the analyzes contemplated here. be able to.

Once the analytical result is obtained as a detectable change, the change is usually measured by passing the test element through an area provided with reflective, translucent, or fluorophotometric equipment. Such devices are designed to apply a flux of energy, such as light. This light is then reflected from the back of the device towards the detector. Analytical results are detected in regions of the element that fall entirely within the region in which such results are produced. The use of reflectance spectrophotometry is
For example, optical interferences from blood cells or residues such as urine sediment remaining on or in the layers of the device or from abnormal urine colors can be effectively avoided. This is useful in situations where: Conventional fluorescence spectrophotometric techniques can also be used. Generally, about
Electromagnetic radiation in the range 200-900nm is
It has been found that this method is useful for such measurements. However, any radiation that can penetrate the reagent layer and quantify the products formed within the device can be used. A variety of calibration methods can be used to provide controls for the analysis. As an example, a sample of a standard solution of the ligand under analysis can be applied in close proximity to the station where the sample solution is placed for differential measurement of the analysis.

The following examples describe experiments performed in developing the invention. While these examples indicate preferred embodiments, they should not be construed as limiting the scope of the invention.

Example 1 Comparison of model systems In order to evaluate the effect of the shielding layer of the present invention, devices were prepared with and without the above-mentioned radiation diffusion/shielding layer between the reagent layer and the support layer. did. The reagent layer in this model system is itself prepared by a detectable response, ie, a reagent solution that fluoresces. Therefore, this example encompasses comparative examples that are not limited to other specific ligands.

Device Preparation Both types of devices were prepared with support layers that all had dimensions of 0.5 cm x 1.0 cm, some of which were polyester and others gel-bound films. Test Devices A and B include a support layer between the radiation diffusing and shielding layer and the reagent layer; Test Device B includes a multi-domain reagent layer. In Test Device C, the reagent layer is between the support layer and the radiation diffusing/shielding layer. Test device D has a radiation diffusing/shielding layer between the reagent layer and the support layer and is an example of the invention.

Test device A was prepared by forming a reagent layer film of the agarose composition on one side of the support layer and applying the TiO ₂ pigment composition to the other side to form a radiation diffusion/shielding layer. The agarose composition has the following formulation.

Ingredients Agarose 1.5g _Triton
A trademark for a series of man-made organic surfactants sold by Rohm & Hass, Philadelphia, PA. Visine is N,N'-bis-(2
-Hydroxyethyl)-glycine. _TiO2
The pigment composition is formed as follows: Ingredient quantities TiO ₂ 2.5 g PVP 2.5 g Chloroform 44.5 g Dioctyl phthalate 0.5 g Preparation of test device B consists of applying a polyvinylpyrrolidone (PVP) composition to the surface in contact with the support layer. As mentioned above, it is the same as that of test device A, except that it is attached as a film to the opposite agarose film surface. This PVP composition forms a film, which is combined with an agarose film to form a multi-domain reagent layer. It was made as follows: Ingredients PVP 5.0 g Chloroform 45.0 g Test device C was prepared by forming a reagent layer of an agarose composition on one side of the support layer. The agarose composition was the same as that used in Test Devices A and B. Using the same TiO ₂ composition used in Test Devices A and B, a film was formed on the side of the agarose film opposite to the side that contacted the support layer.

Test device D is the same as that used in other test devices.
A TiO ₂ pigment composition is applied onto the support layer to form a radiation-diffusing and shielding layer, and then the same agarose composition used in the other test devices is applied to the side opposite to the side in contact with the support layer. It was prepared by coating it on top of the agarose layer.

In addition, 0.5 x 1.0 cm Whatman 31ET paper (Whatman, Inc., Clifton.NJ07014) and reflective Mylar polyester (3M Company, St.
Paul, MN55144) were laminated together to form Test Device E. No reagent is present in the formed test device.

Analysis method The test devices prepared as described above were
The test device was inserted into a mechanical holding fixture suitable for horizontal positioning within the fluorometer. 7-hydroxy-coumarin-3-carbooxanilide (HCC) 30μ
One drop of an aqueous solution of was placed on each test device immediately before placing it on the fluorometer fixture. Each HCC solution used was 0.2~
The concentration ranged from 2.0 μM.

The fluorometer was adjusted to produce an excitation light source with a wavelength of 405 nm across the surface of the device at 90 degrees from the plane of the surface, and the light emitted at a wavelength of 450 nm was measured. Front photometry of fluorescence was performed at an angle of 90° from the horizontal plane of the element surface. The fluorescence of each sample was then measured.

Results The range R and the signal to background ratio (S/B) for each HCC sample are calculated using Equations 1 and 2, respectively: (1) R=Fm−Fb (2) S/B=Fm/Fb (where , Fb and Fm are the fluorescence signals for the blank and HCC solution, respectively).

The data obtained using test devices A and B are shown in both FIGS. 1 and 2 by the dashed curves. The data obtained using test tools C and D are as follows:
This is shown in both FIGS. 1 and 2 by the curve drawn as a solid line. The data obtained using test device E are shown in both FIGS. 1 and 2 by curves drawn with phantom lines.

Figure 1 shows a plot of range as a function of HCC concentration for each system. Apart from Whattmann 31ET (Test Device E), two separate responses were observed for the TiO ₂ system. Those with TiO ₂ below the support (test fixtures A and B) are better than those with TiO ₂ on the top side of the support (test fixtures C and D), whether gelbond or polyester. Whether gel bonded or polyester,
Shows low range in contact with HCC. Whatman 31ET (Test E) shows a better range than the TiO ₂ system.

When the signal/background (S/B) ratio is plotted against HCC concentration as shown in Figure 2, the difference between test devices A and B on the one hand and test devices C and D on the other hand. The same phenomenon is observed. H.C.C.
TiO ₂ on top of the support blocking the solution-receiving layer
Those equipped with this exhibit a large S/B ratio. The one with _TiO2 under the support shows a smaller ratio,
And the Whatman 31ET (test device E) is in the middle.

Example 2 Substrate-labeled fluorescent immunoassay device for theophylline Theophylline [1,3-dimethylxanthine,
[See Merck Index, 9th edition, p. 1196 (1976)] is a drug useful in the treatment of asthma. In most patients, the therapeutic range of serum concentrations is between 10 and
It is at 20 μg/ml, but the toxicity is almost certain.
Appears at blood concentrations exceeding 35 μg/ml.

Preparation of complex A β-galactosyl-umbelliferon-labeled theophylline (β-GUT) complex was prepared according to the reaction formula shown in FIG. This synthetic route is
-[3-(7-β-galactosilmarin-3-
carboxamide) propyl] theophylline (4), n
The following method for preparing =3 was exemplified.

8-(3-aminopropyl)theophylline (2) 8-(3-carboxypropyl)theophylline
(1) [Cook et al., Ros.Commun.Chem.Path.
Pharmacol. 13(3): 497-505 (1976)], 20 ml of chloroform, and 3 ml of concentrated sulfuric acid were stirred at 50° C. under an argon atmosphere. to this,
1.3 g of solid sodium azide was added in portions over 90 minutes [Organic Reactions 47:28
(1967)]. The reaction mixture was cooled and the solvent was removed under reduced pressure. The residue was mixed with enough sodium carbonate solution to bring the pH to 7.5. 10 g Celite (Fisher Scientific Co., Pittsburgh,
Pennsylvania) was added and the water was allowed to evaporate. Celite is a trademark for certain diatomaceous earth products. Impregnated Celite with ethanol-1 moral aqueous triethylammonium bicarbonate 9:1
200 g of silica gel prepared in (V:V) (E.
Merck Co., Darmstadt, West Germany) at the top of the column. Elute the column with this solvent
Fractions of 15 ml were collected. Fractions No. 171-225 were combined and evaporated to obtain 500 mg of white powder. This is ammonium-type CM-Sephadex (Pharmacia Fine Chemicals, Piscataway,
Again, using the column from New Jersey, USA),
Chromatographed and eluted with 0.5 molar ammonium bicarbonate. Floor volume is 3cm x 50cm
10 ml fractions were collected. Fractions No. 65-110 were combined and evaporated to obtain 250 mg of white solid. It was dissolved in dilute hydrochloric acid and evaporated again.

The residue was recrystallized from methanol to obtain 90 mg of hydrochloride (yield: 3%) as light brown needle-shaped crystals that did not melt below 300°C.

Analysis: Calculated value as C ₁₀ H ₁₆ N ₅ ClO ₂ : C,
43.88; H, 5.89; N, 25.59 Actual value: C, 43.77; H, 5.88; N,
25.46 Infrared absorption spectrum (KCl): 1695cm ^-1
and 1655 cm ^-1 (amide carbonyl).

8-[2-(7-β-galactosilmarine-
3-Carboxamido)propyl]-theophylline (4) 24 g potassium hydroxide, 80 ml water, 240 ml methanol and 20 g (0.035 mmol) ethyl 7-
β-galactosylmarin-3-carboxylate (Burd et al, Clin.Chem.23:1402 (1977))
A reaction mixture was prepared containing: The reaction mixture was stirred for 15 hours at 50°C. Upon cooling, methanol was removed under reduced pressure. This concentrated aqueous solution was diluted with concentrated hydrochloric acid to pH2.0.
made acidic. Collect the white precipitate, wash with cold water,
Recrystallized from hot water. The crystals were collected, washed with acetone, and dried at 80°C for 1 hour. as white crystals
12 g of 7-β-galactosilmarin-3-carboxylic acid was obtained. Melting point 250-255℃.

A mixture of 1.45 g (0.004 mol) of 7-β-galactosilmarin-3-carboxylic acid, 404 mg (0.004 mol) of triethylamine, and 40 ml of dry dimethylformamide was cooled to −10° C. with stirring under an argon atmosphere. This is supplemented with 546 ml (0.004 mol) of isobutyl chloroformate (Aldrich
Chemical Co., Milwaukee, Wisconsin) to form mixed anhydride (3). 10 minutes later, another 404
mg of triethylamine and 949 mg (0.004 mol) of 8
-(3-aminopropyl)theophylline (2) was added to the flask. After stirring at −10° C. for 30 minutes, the reaction mixture was warmed to room temperature. It was combined with 10 g of silica gel and DMF was removed under high vacuum. Place the impregnated silica gel on top of a 170 g silica gel column and elute the column with absolute ethanol for 15 min.
ml fractions were collected. Fractions No. 41-475 were combined and evaporated to give 545 mg of a yellow solid. After dissolving it in water, it was filtered and concentrated to a volume of 20 ml. A small amount of precipitate formed was discarded. Pour the liquid into Cephadex LH-20 Gel (Pharmacia Fine
Chemicals, Piscataway, New Jersey) 2.5
It was purified by chromatography using a cm x 57 cm column, eluted with water, and 15 ml fractions were collected.
Sephadex is a trade name for a hydrophilic, insoluble chromatographic medium made from cross-linked dextran. Fractions No. 18-23 were combined and evaporated, and the residue was recrystallized from water to give 55 mg of the labeled complex (4), pale yellow in color and with a melting point of 190-192°C.
(Yield 2%) was obtained.

Analysis: Calculated as C ₂₆ H ₂₉ N ₅ O ₁₁ : C,
53.15; H, 4.98; N, 11.92.

Actual value: C, 52.65; H, 5.01; N,
11.80 The synthesis described above of the β-galactosylmarin-theophylline conjugate (4), n = 3, is performed using the starting material 8-
(3-carboxypropyl)theophylline (1), n
By replacing =3 with a suitable 8-(ω-carboxyalkyl)theophylline as follows, it can be modified to obtain labeling conjugates for n=2-6.

nAlkylene 2 Ethylene 4 Butylene 5 Pentylene 6 Preparation of hexylene antiserum Res.Comm.Chem.Path.Pharmacol.13:497-
Antisera were collected from rabbits immunized with theophylline immunoconjugates prepared as described by Cook et al., 505 (1976).

Preparation of the device The device prepared for use in these experiments was
It had the form shown in FIG. The support layer 2 is made of polystyrene. The impermeable radiation-diffusing and shielding layer 4 has the following composition: 10 g of styrene/maleic anhydride (50/50 copolymer, molecular weight 50000) 6 g polyethylene glycol (molecular weight 1000) 10 g titanium dioxide 34 g acetone Consists of.

This composition was applied to a wet thickness of 0.02 inch onto polystyrene support layer 2 and then dried at room temperature.

The gelatin undercoat layer 6 is an opaque radiation-diffusing layer.
One region of the multi-region reagent layer 12 is formed in close contact with the surface of the shielding layer 4 . The subbing layer consists of a composition having the following composition: 5 g of gelatin 45 g of 0.1 M bicine buffer (PH1 1.1).

This composition was coated over layer 4 at a wet thickness of 0.005 inch and then dried at 37°C.

Antibody and enzyme content 8 was prepared using the following composition: 4% (W/
_V ) Agarose LGT 1.5ml 10% Triton It was prepared from a composition having

Agarose LGT is an agarose material that has a low grilling temperature below 40°C and is manufactured by Research Products Division of Miles Laboratories, Inc.
Laboratories, Inc.). , P-O Boxes
2000 (POBox 2000), Elkhart,
Sold by Indiana 46515. Apply this composition in a wet thickness over layer 6.
A 0.02 inch coating was applied and dried at 37°C. It is the middle region of the multi-region reagent layer 12.

The composition of the composite-containing layer 10 is as follows: β-GUT composite 6% (W/V) PVP (molecular weight) in 69.2 μ CHCl ₃ (1.93 ml)
360000) 2.90g Triton X-100 5μ.

This composition was applied to layer 8 to a wet thickness of 0.005 inch and dried at room temperature. It is the top region of the multi-domain reagent layer 12. The exposed top surface of layer 10 is the surface to which the sample is applied and readings taken.

Analysis method A 35μ fraction of the drug solution was dropped onto the exposed surface of the analytical element prepared as described above.

The fluorescence generated at room temperature was measured for 5 minutes in a fluorometer equipped with a suitable mechanical holder for horizontal positioning of the analytical element. The fluorometer was adjusted to provide an excitation light source at 405 nm, which struck the table at 90°, and to detect the emitted light at a 450 nm wave. Fluorescent front metering was performed at 90° from the pad.

The concentration ranges analyzed were as follows:

Range Theophylline Therapeutic range 10-20 μg/ml Dose-response range investigated 0-41 μg/ml The dose-response range investigated covers the therapeutic range.

Results The data obtained with the method described above are illustrated graphically by FIG. The classification unit is expressed in terms of millibottle (mV). A 1 mm bottle is 1/1000th of 1 volt. A dose-response curve, FIG. 6, was constructed from the data shown in FIG. 5 at tertile points.

Discussion The data obtained demonstrate that the integrated analytical element prepared according to the present invention provides a quantitatively detectable signal responsive to the concentration range of theophylline present. Increasing the concentration of theophylline leads to increased drug dependence in the fluorescence of the respective analytical element.

[Brief explanation of the drawing]

1-2 are graphs of data obtained from the experiment described in Example 1. FIG. 3 is a representation of the method used to prepare the conjugate used in Example 2. FIG. 4 is a cross-sectional view of a theophylline analytical element prepared as described in Example 2. Figures 5-6 are
2 is a graph of data obtained from the experiment described in Example 2.

Claims (1)

[Scope of Claims] 1. A reagent layer containing a reagent that provides a detectable response in response to a ligand in a sample or the ability to bind to a ligand; a radiation scattering/shielding layer; and a support layer. A multilayer analytical element for detecting a ligand or the ability to bind to a ligand in a liquid sample, comprising: (a) a radiation scattering/shielding layer between the reagent layer and the support layer; (b) impermeable to the ligand, the reagent present in the reagent layer, and the products of their interaction; and (c) impermeable to the ligand, the reagent present in the reagent layer, and A multilayer analytical element characterized by being inert to the products of their interaction. 2. The multilayer analytical element according to claim 1, wherein the radiation scattering/shielding layer is opaque, relatively thin, and contains a white or light-colored pigment. 3. The multilayer analytical element according to claim 2, wherein the pigment is titanium dioxide. 4. The multilayer analytical element according to claim 2, wherein the pigment is barium sulfate. 5. The multilayer analytical element according to claim 2, wherein the pigment is zinc oxide. 6. The multilayer analytical element according to claim 2, wherein the pigment is magnesium oxide. 7. The multilayer analytical element according to claim 2, wherein the pigment is zirconium dioxide. 8. The multilayer analytical element according to claim 2, wherein the radiation scattering/shielding layer is made of an anhydride resin. 9 Radiation scattering/shielding layer has a thickness of 0.0002 to 0.02
The multilayer analytical element according to claim 2, which is inches. 10. The multilayer analytical element according to claim 2, wherein the pigment is uniformly dispersed throughout the radiation scattering/shielding layer. 11. Claim 1, wherein the reagent layer includes a reagent for a homogeneous specific binding assay system that produces a detectable response that is a function of the presence of a ligand or the ability to bind to the ligand in the sample. Multilayer analysis element described in Section 1. 12. The multilayer analytical element according to claim 11, wherein the homogeneous specific binding analysis system includes a label involved in an enzyme reaction. 13. The multilayer analytical element according to claim 12, wherein the label is a substrate for an enzyme. 14. The multilayer analytical element according to claim 13, wherein the enzyme acts on the substrate label to produce a detectable product. 15. The multilayer analytical element according to claim 12, wherein the label is a prosthetic group (compound group) of an enzyme. 16. The multilayer analytical element according to claim 15, wherein the prosthetic group can be combined with the apoenzyme to form an enzyme. 17. The multilayer analytical element according to claim 12, wherein the label is an enzyme. 18 The reagent layer contains (i) an antibody that binds to the ligand, (ii) a complex of the ligand or its binding analog and a label, and (iii) interacts with the label and binds to the antibody. The reagent composition includes a sensing system that produces a different, detectable response when bound than when unbound, such that the detectable response is a function of the presence of the ligand in the liquid sample. 2. A multilayer analysis element according to claim 1, comprising a material. 19. The multilayer analytical element according to claim 18, wherein the detection system participates in an enzymatic chemical reaction involving a label. 20. The multilayer analytical element according to claim 19, wherein the label is a substrate for an enzyme and the detection system includes the enzyme. 21. The multilayer analytical element according to claim 19, wherein the label is a prosthetic group of an enzyme, and the detection system includes an apoenzyme that together with the compound group forms the enzyme. 22. The multilayer analytical element according to claim 21, wherein the detection system further includes an indicator for enzyme activity. 23. The multilayer analytical element according to claim 21, wherein the label is flavin adenine dinucleotide and the apoenzyme is apoglucose oxidase. 24. The multilayer analytical element according to claim 21, wherein the detection system further includes an indicator for glucose oxidase activity. 25. The multilayer analytical element according to claim 24, wherein the indicator contains a substance that reacts with color to glucose, peroxidase, and hydrogen peroxide. 26. The multilayer analytical element according to claim 19, wherein the label is an enzyme and the detection system includes an indicator of enzyme activity. 27. A reagent for a homogeneous specific binding assay system in which the reagent layer (a) produces a detectable response that is a function of the presence of a ligand or the ability to bind to the ligand in the sample; and (b) a solid carrier containing a reagent; a radiation scattering/shielding layer comprising a pigment encapsulated in an anhydride resin; the layer: (a) interposed between the reagent layer and the support layer; (b) impermeable to the ligand, the reagent of the reagent layer, and the products of their interaction; and (c) impermeable to the ligand, the reagent of the reagent layer, and the products of their interaction. A multilayer analytical element according to claim 1, which is inert against. 28. In a liquid sample of the type having a reagent layer, a radiation scattering/shielding layer, and a support layer containing a reagent that provides a detectable response in response to the ligand or the ability to bind to the ligand in the sample. A method for manufacturing a multilayer analytical element for detecting a ligand or the ability to bind to a ligand, the method comprising: (1) a surface of a support layer; impermeable to the reagents and the products of their interaction; and (b) inert to the ligand, the reagents of the reagent layer, and the products of their interaction. A method comprising the steps of: (2) adhering the surface of the reagent layer to the opposite surface of the radiation scattering/shielding layer.