JPH0560059B2 - - Google Patents

Description

[Detailed description of the invention]

[Detailed Description of the Invention] The present invention relates to an apparatus for quantifying an analyte in a sample suspected of containing the analyte, and a method for quantifying the analyte. There is continued interest in developing new, simple, and rapid techniques for detecting and measuring the presence of analytes in samples. The analyte can be any of a wide variety of substances, such as drugs, naturally occurring physiological compounds, contaminants, chemicals, impurities, and the like. In many cases, especially in the case of certain physiologically active substances, the speed of measurement is important. In other situations, simplicity is the primary consideration. One simple and rapid technique that has found wide application generally involves a solid rod or film that can be immersed into a sample and subsequently emitted a signal based on the amount of analyte in the original sample. This is the "dip stick" method. There are many devices available for measuring the signal of a compound bound to a solid surface, such as absorption, reflection or fluorescence. Moreover, the stick dipping method is easy to handle, move, separate, etc. Although convenient, such techniques are very sensitive to development time, temperature, interfering factors, reagent stability, and other conditions that can affect the amount of signal observed. For quantitative analysis, where the observed signal is compared to a standard, it is necessary to carefully control the test conditions and match the conditions of the standard. However, such careful control reduces the simplicity of the dip rod method, increases the time and expense of performing the analysis, and increases the instability of the results. Furthermore, the presence of interfering factors in the sample and the instability of the reagents are difficult to overcome even with the greatest care. It is therefore desirable to develop new analytical methods that provide accurate detection of analytes in samples and are not very sensitive to development time, temperature, interfering factors in the sample, stability of reagents, etc. A patent related to the use of various immobilized reagents and different types of test strips is U.S. Patent No. 3,993,451.
No. 4038485, No. 4046514, No. 4129417,
Nos. 4133639 and 4160008; and German Published Patent Application No. 2636244. Patents disclosing various methods involving separation of bound and unbound antigen include:
U.S. Patent No. Re.29169, No. 3949064, No.
No. 3984533, No. 3985867, No. 4020151, No.
No. 4039652, No. 4067959, No. 4108972, No.
Including No. 4145406 and No. 4168146. The present invention provides devices and methods for quantifying the presence of an analyte in a sample suspected of containing the analyte. The method and apparatus include first and second surfaces referred to as measurement and calibration surfaces. Each preferably includes a signal generating system having at least one catalyst and one substrate. Both systems are substantially identical and include at least one common member of the signal generating system. The measurement surface contains a conjugate bound to the surface by means of generating a specifically binding pair complex, while the conjugate is a member of a signal generating system ("sps") that binds to one member of the specific binding pair. "Members"). The amount of conjugate bound to the surface is related to the amount of analyte in the assay medium. The calibration surface has sps members bound to the surface, either covalently or non-covalently, directly or through the creation of specific binding pair complexes. In this case, the specific binding pair is different from the binding pair of the measurement surface. By comparing the amount of signal-generating compound on each surface, it can be determined whether the amount of analyte is greater or less than the predetermined amount indicated by the signal generated from the calibration surface. According to the present invention, a method and apparatus are provided for quantifying an analyte in a sample suspected of containing the analyte by jointly introducing at least two different surfaces into a liquid analytical medium. Ru. One surface is then called the measurement surface and the other surface is referred to as the calibration surface. The analysis is preferably at least 1
It involves the use of a signal generating system having one catalyst, usually an enzyme, and at least one substrate. To amplify the signal, it is desirable that there be multiple signal generating events for each binding event of an analyte to its cognate binding partner. This plurality may exist as a result of dynamic and static systems. In dynamic systems, a catalyst is used, usually an enzyme, and the signal generating system contains at least one substrate, usually a coenzyme. In this case either the enzyme or the substrate may be bound to a specific binding partner. If the substrate cannot be cycled, usually multiple substrate molecules will bind to the hub to provide multiple substrate molecules for a single binding event. Alternatively, multiple non-catalytic molecules that provide a detectable signal can be attached. Such molecules would normally be detectable spectroscopically. Although dyes that absorb light may be useful in some cases, fluorescence and chemiluminescence are used to generate signals that can be more accurately measured with commonly existing equipment. The amount of sps member present as a specific binding partner that binds to the measurement surface through the sps member conjugate and specific binding pair complex is related to the amount of analyte present in the medium. The amount of sps member bound to the calibration surface during analysis will be such as to give an advantageous range of signal amounts for at least one, and usually one, predetermined analyte concentration. on the calibration surface
sps members may be present as a result of covalent or non-covalent binding prior to or during immersion into the analysis solution containing the sample. However, in the case of non-catalytic sps members, the binding is non-covalent and subsequent immersion into the analytical solution. The method and apparatus simultaneously calibrate the analytical system during each individual test. The signal generation system is associated with the generation of a detectable signal on two surfaces and is subjected to identical conditions with respect to many conditions that affect the observed signal. Therefore, variations in the detectable signal due to changes in conditions, endogenous substances in the sample, etc. affect the detectable signal generation on the two surfaces in parallel, and the signal amount on the calibration surface is equal to the signal amount on the measurement surface. can serve as a standard for evaluation. The invention relies on the use of two surfaces each employing a signal generating system that produces a detectable product. The amount of sps member bound to the measurement surface is related to the amount of analyte in the medium. The formation of a detectable product that generates a signal at the surface is
It will be directly related to the amount of sps members. In contrast, the amount of sps member bound to the calibration surface will not depend solely on, and is usually independent of, the amount of analyte in the medium. sps present relative to the calibration surface
The amount of analyte will normally be independent of the amount of analyte in the assay medium, even when not pre-bound to the calibration surface. Once an sps-membered molecule binds to a surface, the signal-generating systems at the two surfaces are exposed to the same environment, and therefore the generation of a detectable signal at the calibration surface is a qualitative indication of the concentration of analyte in the medium. or can be used as a reference for quantitative analysis. For the most part, in the case of macroscopic analysis, the calibration surface is not used to analyze the specific concentration, but rather the presence or absence of the intended analyte in an amount greater than a predetermined amount. It will be used to decide whether or not to When performing an analysis, many measurement procedures and combinations can be used to vary the substances bound to the two surfaces. In relation to the two surfaces,
It should be understood that more than two surfaces may be used, ie one or both of the measurement and calibration surfaces may be present in plurality in the device. The calibration surface involves initial binding of sps members to the surface, except for non-catalytic systems, when a predetermined amount of catalyst is bound to the surface. Alternatively, specific binding partners can be used for calibration surfaces that bind sps members or specific binding partners that bind sps members. Such a specific binding partner binds to a determinant site of the analyte other than the determinant site involved in binding to a specific binding partner cognate to the analyte. The measurement and calibration surfaces can be made from any convenient material and have any convenient construction. However, the surface may substantially retain its structural integrity during analysis. The two surfaces are typically juxtaposed such that they are subjected to the same environmental conditions to ensure that the sps members on both surfaces are responsive to substantially the same type of environmental conditions. Various signal generation systems can be used that are readily applicable to a given subject or a given condition. In each case, the signal generating system contains at least one functional entity capable of giving a detectable signal, preferably at least one catalyst, usually at least one enzyme, and at least one substrate with the catalyst sps. Will. In this case, the sps member will bind to the specific binding partner to produce a conjugate that binds at least to the measurement surface, and may bind to both the measurement and calibration surfaces. A device containing two surfaces will be brought into contact with one or more solutions in which the analyte and conjugate have had the opportunity to bind to the measurement surface and, where appropriate, to the calibration surface. In the case of a catalytic system, the two surfaces are optionally introduced into a reagent solution containing the necessary substrates and cofactors for the signal generating system. However, in some situations, for example when using two catalysts, all of the signal generating systems can be combined together with the sample in a single analysis medium. After a predetermined time, the surface is removed from the developer solution and the detectable signals on the measurement and calibration surfaces are compared. Let us now describe the invention in more detail. However, before describing the invention in detail, a number of terms will be defined. Definition Analyte - mono- or poly- which may be a ligand
epitopic, usually a compound or composition to be measured that is an antigen or an adherent. The compound or compounds share at least one common epitopic or determinant point or receptor. Specific binding pair ("mip") - two different molecules that have regions on their surfaces or cavities in which one of the molecules specifically binds to a special spatial and polar organization of the other molecule. The members of a specific binding pair are referred to as the ligand and receptor (anti-ligand). These are commonly immunological pairs, but other specific binding pairs such as biotin-avidin, hormone-hormone receptor, etc. are not immunological pairs. Homologous or complementary substrates are ligands and acceptors, while homologous substrates are either distinct, eg labeled, ligands or acceptors in certain situations. An organic compound in which a ligand-acceptor occurs naturally or can be prepared. Receptor (anticoordinator) - A compound or composition capable of recognizing a special spatial and polar organization of a molecule, ie, an epitopic point or decision point. Examples of receptors include naturally occurring receptors such as globulins that bind thyroxy, antibodies, enzymes, Fab fragments, lectins, nucleic acids, and the like. Coordinator homologue - A modified ligand that can compete with a homologue ligand for an acceptor. This modification provides a means for attaching the ligand analog to other molecules. Coordinator homologs will normally differ from the ligand by replacing more than one hydrogen with a bond linking the ligand homolog to the center or label, but this is not necessary. . Poly(coordination homolog) - Multiple ligands or ligand homologues that are usually covalently bonded together to a central core. The central core is usually a polyfunctional, usually polymeric material having multiple functional groups such as hydroxy, amino, mercapto, ethylene groups, etc. as points of attachment. This central core is usually water soluble or at least dispersible and usually at least 35,000 daltons;
Generally it will not exceed about 600,000 Daltons. Examples of cores include polysaccharides, polypeptides such as proteins, nucleic acids, ion exchange resins, and the like. Surfaces - The measurement and calibration surfaces are each non-dispersive, typically present on a common support, and have a surface area of at least about 50 μm ² , generally larger, and often at least 1 μm 2 .
mm ² , especially about 0.5 cm ² or less, and which is insoluble in water and provides the necessary properties for the binding of a mip and a detectable signal-generating compound to give the desired amount of signal. The substance may be Desirably the surface is gelatinous, permeable, hygroscopic, porous, or has a rough or irregular structure which may generally be channels or the like having a substantial void volume compared to the total volume. Probably. Depending on the nature of the signal-generating compound to be detected, the surface may be absorbing or non-absorbing, preferably weakly absorbing or non-absorbing. The surface may be transparent or opaque and may be a single material or multiple materials, mixtures or laminates. A wide variety of materials and forms can be used. The surface can substantially retain its integrity under the conditions of analysis, such that substances bound to the surface remain bound to the surface and do not diffuse into solution. It is desirable that the underlying structure of both the measurement and calibration surfaces be substantially the same. Catalyst - binding - mip - a catalyst conjugated to mip, usually an enzyme. The catalyst is a member of the signal generating system and the mips are selected to bind to the measurement surface depending on the particular step sequence. Signal Generating System (“SPS”) - The signal generating system may be catalytic or non-catalytic, preferably catalytic, and more preferably has enzymatic catalysis; desirably the product of one enzyme is the product of the other enzyme. Two enzymes are used that are substrates; in addition, the catalytic signal generating system will include at least one substrate, and, where appropriate, a coenzyme. The signal generating system emits a detectable signal at the measurement surface that is related to the amount of analyte present in the medium, while at the calibration surface at least one ligand-receptor pair, e.g. body or antibodies,
Contains enzymes and substrates or enzymes. The signal generating system emits a detectable signal at the calibration surface, in whole or in part, by a "signal generating compound" that binds to the surface and emits a detectable signal. The amount of detectable signal at the calibration surface is independent of the amount of analyte and depends on at least one factor. Other substances that can be included in the signal generating system are light for the fluorophore, reactant for chemiluminescence and a capture agent for the intermediate product if at least two enzymes are used. For simplicity, members of the signal generation system will be referred to as "sps members." The sps member may be a fluorophore, a chemiluminescent substance, an enzyme, or a substrate where the term substrate includes a coenzyme. Method This analysis is performed in an aqueous region or medium. However, the final analytical medium does not require prior addition and incubation of each reactant or combination of reactants, removal of the surface from the aqueous medium, and transfer to a different aqueous medium with one or more reactants. or a combination thereof. This method removes the unbound labeled conjugate from
Although it is not necessary to separate one or both surfaces from those bound through the mip complex, in many processes a developer solution that is substantially free of unbound catalyst will be used. The various media involved in the analysis consist of liquid and solid phases that define the measurement and calibration "surface". For analysis, the surface is contacted in an aqueous medium with a sample, a signal generating system, and an auxiliary substance, either simultaneously or stepwise, to obtain a detectable signal associated with the surface. The detectable signal at the measurement surface is related to the amount of labeled conjugate bound to that surface, which corresponds to the amount of analyte in the sample. Depending on the nature of the signal generating system and the desired method of detecting the signal, the surface can be read in the analysis medium or separately from the analysis medium. When carrying out the analysis, an aqueous medium will normally be used. Oxygenated organic solvents having 1 to 6 carbon atoms, and usually 1 to 4 carbon atoms, including other polar solvents, common alcohols, ethers, etc., may also be included. Normally these co-solvents will be present at up to about 40% by weight, even up to about 20% by weight. The PH for the medium will normally range from about 4 to 11, more usually from about 5 to 10, preferably from about 6.5 to 9.5. The PH is chosen to optimize the signal generation system while maintaining a significant amount of specific binding by the receptor. In some cases a compromise will be made between these two considerations. desired
Various buffers can be used to achieve and maintain the PH during analysis. Examples of buffering agents include borates, phosphates, carbonates, Tris, barbital, and the like. The particular buffer used is not critical to the invention, but some buffers may be more suitable than others in a particular analysis. Moderate temperatures are usually used to perform the analysis. A constant temperature during the measurement period is generally not necessary, but rapid and large fluctuations are undesirable. The temperature for analysis is generally about 10-50°C, more commonly about 15-45°C.
It is ℃. The concentration of the analyte that can be analyzed is generally about 10 ^-4 ~
10 ^-15 M, more commonly around 10 ^-5 to ^{10 -13} M. Depending on whether the analysis is qualitative, semi-quantitative or quantitative, the detection method used and the concentration of the analyte in question will usually determine the concentration of the other reagents. The concentrations of the various reagents will vary depending on the process procedure used, the nature of the analyte, the mips bound to the surface and the catalyst bound to the catalyst.
varies widely depending on mip, required analytical sensitivity, etc. In some cases one or the other of the mips will be used in large excess, while in other steps the sensitivity of the assay will be responsive to changes in the ratio of mips. When performing the calibrated analysis of the present invention, both the measurement and calibration surfaces need to be contacted simultaneously with the sample and signal generation system. It is also desirable that both surfaces be located close to each other to minimize differences that may be due to local variations in the medium. Conveniently, this may be achieved by placing both surfaces on a common rod or support.
However, when carrying out the method of the invention, it is not necessary to place the surfaces on a common support, but to immerse the surfaces in the various components of the signal-generating system,
It is only necessary that they be taken out at the same time and for the same length of time. Otherwise, the surfaces may be handled independently without adversely affecting the performance of the analysis. A common support for the surface is conveniently a rod or plastic film as used in conventional rod dipping. The exact nature and dimensions of such a dipping rod are not critical and are selected to be compatible with other factors of the analytical system, typically the dimensions of the various reagent containers. Both surfaces are located at one end of a long dipping rod;
It is desirable that it can be easily immersed into a relatively small volume of sample, typically 100 μl to 2 ml. Positioning the surfaces next to each other facilitates visual comparison of the surfaces to make a final determination. The surface may be located vertically or horizontally. As already indicated, more than two surfaces can be used, including multiple measurement surfaces and multiple calibration surfaces, one or both. For example, multiple analytes may be analyzed simultaneously and/or multiple calibration surfaces may be arranged to provide different calibration surfaces or even quantitative results in conjunction with each measurement surface for a different analyte. . Various process procedures using one or more solutions are used. Contacting the solution includes stirring or contacting. It also includes an incubation step, which can generally vary from about 0.5 minutes to 1 hour, more usually from about 2 minutes to 30 minutes. The process procedure depends on the signal generation system, the properties of the specimen,
and will vary with considerations of economy, efficiency and accuracy. The reagents of the signal generating system may be provided as separate solutions or may be dissolved and non-conjugated to the surface and diffused into the sample solution. If the signal generating system reagents are provided separately, all of the reagents can be combined with the sample; alternatively, one or more sps members can be combined with the sample and then the remaining members of the signal generating system can be combined. It may be combined with a second solution. For example, the enzyme-label-mip can be combined with the sample solution. The substrate and any coenzymes are then distributed between the sample solution and the second solution, referred to as the developer solution, or any remaining sps members added as a second solution. Alternatively, sps members can dissolve and bind to surfaces in a non-conjugative manner and rapidly dissolve into the sample solution upon contact with the sample solution. If multiple solutions are used, the surface will be transferred from solution to solution. Normally intermediate wash steps will not be required to remove incidental specific binding. The methods and systems of the invention can be used for the analysis of any analyte by using a label that emits a detectable signal. Competitive and non-competitive process procedures can be used; the analyte and sps-labeled analyte may compete for the congener mip on the measurement surface, or they may bind sequentially. Or if the analyte has multiple binding points,
It consists of mip and sps-labels bound to the measurement surface.
It can serve as a bridge between mips. By varying the various mips on the surface and in the sps conjugate, as well as the number of solutions with which the surface is in contact and the members of the signal generation system, the degree of quantification desired, user artifacts, and equipment present can be adjusted. The process procedure can vary widely depending on the conditions. Furthermore, some or all of the same considerations will affect the nature of the calibration surface and the manner in which the sps member is coupled to the calibration surface. The signal generating system may be non-catalytic or catalytic. Non-catalytic systems contain a plurality of signal-generating labels, such as fluorophores or chemiluminescent substances, attached to the center or particle, allowing a relatively large signal to be obtained from a single binding. The mip, selected according to the nature of the analyte, will be present in the center or bound to the particle. In the case of adherent analytes, antibodies against the adherent (anti-adherent) may be bound to the measurement surface. This measurement surface is then immersed in the sample solution.
The analyte binds to and fills the area at the binding point. At this time, the particle-adherent conjugate binds to the remaining anti-adherent points. Alternatively, the particle-adhesive conjugate may combine with the sample solution and compete with the analyte for binding points present on the measurement surface. The catalytic system may include a catalyst on a substrate bound to a mip. When substrates are bound to mips, the hub is usually abundant, as in noncatalytic systems, and contains multiple substrates and one or more mips.
It will be used with. If one of the substrates is a coenzyme, the coenzyme may be recycled by using a two enzyme system. In this case, the result is the production of multiple signal-generating compounds due to the presence of a single coenzyme. These systems are well reported in the literature. When the substrate is not cycled, it will normally be a precursor to the fluorophore or will give rise to chemiluminescence. In a catalytic system comprising a single catalyst, usually an enzyme, bound to mip, the catalyst-bound-mip and analyte are
It can be used in a competitive manner such that the catalyst-bound mip competes against the congener mip simultaneously or sequentially at the measurement surface. That is, a sample containing an analyte may be contacted with a surface in combination with a catalyst-bound-mip, or subsequently a catalyst-bound-mip
can be brought into contact with. In the former case, the catalyst -
Binding-mips are present in relatively limited amounts and compete directly with analyte for mip binding points to the measurement surface. In the latter case, the catalyst-binding-mip fills the binding points remaining after binding of the analyte to the measurement surface;
Therefore, a concentration of the analyte in question can be present in greater excess than in the former situation. mip
After sufficient time for the surface to bond to the measurement surface,
It may be contacted with a suitable substrate containing cofactors, including compounds attributable to the product that will bind to the surface and give a detectable signal. In other embodiments, a combination of catalysts, especially with at least one enzyme, is used. In this case, one of the catalysts produces a product that is a substrate for the other catalyst. This is a preferred embodiment, particularly in the case of two enzymes, in that it minimizes the number of reagent solutions and/or washing requirements and rapidly generates the signal generating compound at the surface. In this embodiment, the measurement surface contains not only the mip but also one of the two enzymes, preferably the first enzyme of the system. Enzyme-linked-mip is preferably the second enzyme in the system. The product of the first enzyme is an essential substrate for the second enzyme, and therefore the various substrates and cofactors required for the signal generation system are first involved without regard to the pre-ripening reaction in aqueous medium. May be used together with enzyme 2. The substrate for the first enzyme repeatedly provides a product that is the substrate for the second enzyme only when associated with the surface. A similar process sequence as described above may be used, but the desirability of using a separate substrate solution from the solution containing the catalyst-label-mip is substantially reduced. A preferred process procedure is to add all members of the signal-generating system to the sample; then introduce the surface into the resulting solution; and allow the reaction to occur for a sufficient period of time to produce a detectable change in signal at the surface. wake up;
and removing the surfaces and then comparing the amount of detectable signal on each of the surfaces as a measure of the amount of analyte in the medium. Alternatively, the sample may be contacted with the analyte first and then the enzyme-conjugated-mip added simultaneously or sequentially with the substrate and cofactor. As mentioned above, excess enzyme-bound-mip can be used in competition. At this time, the analyte first binds to its capture mip on the surface. When the analyte serves as a bridge, especially when different monoclonal antibodies are used at the surface, as opposed to enzyme-linked mips,
Regardless of whether the enzyme-conjugated-mip is added simultaneously with the analyte or sequentially to the analyte, an excess amount of the enzyme-conjugated-mip may be used. The concentrations of substrate and cofactor will preferably be present in such substantial excess that they become the limiting factor with respect to the repetition rate of the enzyme. That is, it will exceed the Michaelis constant for the enzyme. The calibration surface will vary depending on the desired sensitivity and the number of factors to be collimated between the calibration surface and the measurement surface. The calibration surface will be parallel to the measurement surface in at least one and preferably two binding events, ie binding events involving homolog mip or enzyme-substrate (including coenzyme) binding. For non-catalytic signal generation systems,
Unlike mip, which is bound to the measurement surface, let's bind to the calibration surface. Preferably, the mip bound to the calibration surface will be of similar nature to the mip bound to the measurement surface, and the two surfaces will have adherents, antigens or receptors as appropriate. For catalytic signal generation systems, the calibration surface does not need to contain mips. The simplest catalytic calibration surface is a catalyst covalently or non-covalently bound to the surface;
At this time, catalytic activity is imparted at a predetermined value. That is, during analytical measurements, the catalyst is subjected to the same exogenous factors and conditions on the calibration surface as the catalyst bound to the measurement surface. That is, temperature conditions, reagent stability, foreign non-specific interferences, as well as local variations in specific interferences, conditions and concentrations, etc., all affect the production of detectable products similarly on the two surfaces. will be affected. The main sources of variation in the production of signal-generating compounds will therefore be influenced in parallel on the measurement and calibration surfaces. Parallelism can also be further enhanced by forming mip complexes on a calibration surface.
This can be achieved in various ways. One method is
The goal is to provide a receptor for enzyme catalysis that does not significantly affect enzyme activity. enzyme-binding-mip
The amount of enzyme bound to the calibration surface will remain substantially constant if the enzyme is in substantial excess over the amount bound to the measurement surface in the concentration range of the analyte in question. However, the binding of the enzyme to its receptor, particularly antibodies, will be affected in a similar manner as the binding of the analyte to its homolog mip. Further parallelism can be achieved by conjugating the mip differently with the analyte bound to the catalyst.
In the case of coordination analytes, different ligands can be attached to the catalyst. In this case, such different or second ligand may be bound to the same catalyst molecule as the analyte or to a different catalyst molecule. 2
If two ligands are bound to the same catalyst, competition will occur between the measurement and calibration surfaces for catalyst-binding-mip. When catalyst-bound-mip is present in substantial excess, the amount of catalyst-bound-mip that binds to the calibration surface is not significantly affected by the concentration of analyte. In some cases, the catalyst-bound-mip may be present in limited amounts, ie, not in substantial excess over the total binding points present on both the measurement and calibration surfaces. The amount of catalyst bound to the calibration surface will vary with the concentration of analyte. Otherwise, the only variation in the amount of catalyst bound to the calibration surface would be as a result of variations in conditions that affect the formation of mip complexes. If a non-catalytic sps member or substrate label is used, it may have two ligands (an analyte and a calibration ligand) attached to the center or particle. That is, depending on the concentration of the conjugates, the two ligands may be present on the same or different conjugates, and when both ligands are on the same conjugate the conjugates may be present in limited amounts or substantially Competitive or non-competitive formats can be used, in which the compound is present in excessive amounts. A substantial excess herein means that the concentration of the conjugate does not change significantly during the analysis. Typically, the calibration surface will emit a signal substantially independent of the amount of analyte in the analysis medium. Appropriate selection of the components bound to the calibration surface provides a detectable signal that provides a predetermined amount of concentration. The detectable signal from the measurement surface will be influenced in approximately the same way as the generation of the detectable signal at the measurement surface. The detectable signal observed at the calibration surface will therefore define a certain concentration amount of the analyte. When determining the amount of analyte, the amount of signal at the measurement and calibration surfaces can be compared and the contrast used as support for the presence of more than a predetermined amount of analyte. Alternatively, it can be analyzed semi-quantitatively or quantitatively by dividing the amount of signal at the measurement surface by the amount of signal at the calibration surface. In this way, non-specific effects of the analysis medium on signal generation are virtually eliminated. If the mip in the sps member-binding mip is a receptor, having an additional opportunity to attach the acceptor to the calibration surface that binds to the antigenic point on the acceptor portion of the sps member-binding mip. I can do it. If the receptor on the surface is an antibody, an anti-(antibody) may be used which may be attached to a convenient decision point on the antibody. In this method, the sps member-bound mip can act as a receptor in binding to the capture mip bound to the measurement surface and as a hypercoordinator in binding to the capture mip bound to the calibration surface. In many cases it will be desirable for the user of the device of the invention to be able to make as few measurements as possible and as few transfers as possible. That is, the method will allow the measurement of most, if not all, reagents, as well as the measurement of samples. Second, it is desirable to have as few transition operations as possible, not only to increase efficiency and reduce the margin for error, but also to reduce the overall time for testing. In the simplest process, all reagents are applied to the surface or combined in a suitable formulation, conveniently a lyophilized powder formulation. If the reagent is applied to the surface, for example by impregnation, encapsulation, adhesion with a water-soluble adhesive or the like, the device need only be placed in the sample solution. The measurement and calibration surfaces are subjected to the same effect insofar as the velocity of the solution is very high. The lyophilized powder formulation is dissolved in a measured amount of aqueous medium containing the sample. After sufficient time for the solution to become homogeneous, the surface is introduced into the sample solution. For non-catalytic signal generation systems, the surface may only be immersed once by appropriate selection of mips. In the case of a catalytic system, a single formulation can be used in which the signal generating system includes two enzymes, one enzyme producing a product that is a substrate for the other enzyme. If the first enzyme is bound to both the measurement surface and the calibration surface, the second enzyme can be combined with the substrate for the first enzyme without involving a prior ripening reaction. The reason is that the reaction does not occur until the first enzyme produces the necessary substrate for the second enzyme. If only one catalyst is used in the signal generation system, at least two solutions are used, one of which has the substrate for the catalyst and the other solution has the catalyst-binding-mip, so that the surface can be divided into two A reagent solution containing a single substrate is used, either in separate contact with the solution, or the catalyst-bound-mip is solublely attached to the surface. The signal generating compound is associated with an increase or decrease in the observed signal. The signal-generating compound, when generated by a catalyst, binds preferentially to the surface and provides a signal that can be detected visually or by a reflectance or fluorometer. The signal generating compound will usually be substantially insoluble in the medium in which it is produced and will be derived directly or indirectly from the catalyst product. If the signal-generating compound is generated adjacent to the surface with a surface-bound catalyst, the proportion of total signal-generating compound from the solution that results from binding of the signal-generating compound to the surface will be minimized. In many situations, scavengers are desirable. For example, when two enzymes are used in a signal-generating system, the production of signal-generating compounds in the bulk medium can be further reduced if a capture agent for the first enzyme is used in the bulk solution. A capture agent for the signal-generating compound is also useful in the bulk medium if the signal-generating compound is generated in the presence of unbound enzyme-linked mips in the bulk medium. other,
Enzyme inhibitors may be used that selectively inactivate enzymes in the solvent, but are substantially inactive against enzymes bound to the surface. This is accomplished by using reversible or self-destructive inhibitors attached to large porous particles that prohibit access of particle-bound inhibitors to the binding points of surface-bound enzymes. I can do it. In many cases, signal generating compounds will enhance the signal at the surface. However, there is also the possibility of reducing the signal at the surface. For example, if the surface is fluorescent, a quencher can be generated that reduces the fluorescence of the surface. Alternatively, surfaces colored with certain dyes may be used that change the color observed upon precipitation of different dyes onto the surface. Other enzyme combinations may be used in which a first enzyme produces a signal-generating compound and a second enzyme destroys the signal-generating compound or destroys an essential intermediate. For example, glucose oxidase and horseradish peroxidase can be used on the surface to mip carralase.
The more catalase that binds to the surface, the less dye will be produced. That is, the presence of the third enzyme as an enzyme-bound mip causes the enzyme-bound to bind to the surface.
The amount of mip may be associated with reducing the observed production of signal generating compounds. For quantitative analysis, it is advantageous to determine the ratio of the amount of signal on the measurement surface as related to the amount of signal on the calibration surface. In order to quantify the amount of analyte, the ratio of the signals from the measurement surface and the calibration surface can be used to quantify the amount of analyte, i.e. by setting a specific period of time from the start of the production of the signal-generating compound to the end of the further production of the signal-generating compound. can be associated with standard values. If the signal-generating compound is generated at a constant rate, time is not a critical factor as long as a sufficient change in signal occurs on both the measurement and calibration surfaces such that no change in signal is anymore observed at the surface. Do not make it take too long. That is, the ratio produces results that are relatively insensitive to time, temperature, and endogenous interferences. Alternatively, when using two correction surfaces,
The ratio of the signals at these surfaces can be used instead of measuring time. The following are examples of two step sequences using a single enzyme and a combination of enzymes. The analyte is a ligand and the two surfaces have receptors for different ligands. The second ligand is then referred to as the calibration ligand. The enzyme is conjugated to both the analyte cognate and the calibrating ligand. A sample is obtained and dissolved in an aqueous buffer medium to a predetermined volume. A lyophilized mixture containing enzyme-conjugated diligand (analyte and calibrating ligand), stabilizers, excipients, and optional buffers is added to the sample to prepare the analyte and enzyme-conjugated diligand. A solution containing the ligand is applied in an amount sufficient to compete with the analyte for the receptor at the measurement surface, but still bind to the calibration surface to a limited extent. The two surfaces are introduced into the solution at the same time and allowed to stand for a reasonable amount of time. The analyte thereby binds to the measurement surface and the enzyme-bound diligand is distributed between the measurement surface and the calibration surface in relation to the amount of analyte in the analysis medium. After sufficient time for binding, generally about 1 to 10 minutes, the two surfaces are separated from the analysis medium and introduced into a developer solution. After a reasonable period of time for sufficient signal-generating compound to form on the surface, the surfaces are removed and the signals on the two surfaces are visually compared. By suitably selecting the amount of mip on the calibration surface, the signal from the calibration surface is greater than the signal from the measurement surface, for example. For example, if it is dark, it can be seen that the analyte is present in less than a predetermined amount. By measuring the signals from the two surfaces and then determining the ratio, this ratio can be compared against a standard that indicates the actual amount of analyte present. Semi-quantitative results can thus be obtained by visual observation or, if quantitative results are desired, by carefully measuring the signals at the two surfaces and comparing them with standards. The next step sequence uses a combination of enzymes and requires one formulation and one contact of the sample and reagent solution with the surface for generation of the signal generating compound. For comparison purposes with previous sequences, in this case two (enzyme-conjugate-formula) are used, with the same enzyme and two different deposits being referred to as the analyte deposit and the calibration deposit. Use with other adherents. The measurement surface will have antibodies against the analyte deposits, while the calibration surface will have antibodies against the calibration deposits. Although the two adherents should not cross-react, they should desirably be similar in nature so that the formation of each binding complex is similarly susceptible to variations in conditions and endogenous substances. The acceptor on the calibration surface is present in a predetermined amount that produces a signal-generating compound independent of the amount of analyte in the analysis medium. In addition to receptors, the surface will also have a comparable amount of a first enzyme producing a product that is a substrate for the enzyme-bound-coordinator enzyme. The process order is illustrated. The two enzyme-bound-ligands do not compete for receptors on each measurement and calibration surface. Therefore, the amount of enzyme-bound-calibration ligand bound to the calibration surface will not be a function of the amount of analyte in the medium. (enzyme-bond-coordinator)
will not react with the substrate presented to the analytical medium in the absence of surface-bound enzyme products, so all reagents can be combined in a single formulation, which can then be combined with the sample. can be done.
The enzyme-bound-ligand that competes with the analyte for the mip on the measurement surface will reduce the amount of enzyme-bound-ligand bound to the measurement surface in relation to the amount of analyte present in the medium. Exists in limited amounts to change. As mentioned above, the formulation may contain buffers, stabilizers, excipients, etc. in addition to the (enzyme-conjugate-coordinator) and substrate. The formulation can be first dissolved in an aqueous medium to provide a solution with the appropriate concentration of reagents. Take a portion of the solution of a predetermined volume and weigh the sample into the solution. The first enzyme is essential for the enzymatic reaction to proceed. The surface is introduced into the solution for a sufficient period of time for signal-generating compounds to form on the surface, at which point it is removed and the amount of signal generated is determined by either visual observation of the surface or by appropriate equipment. Quantify. Since the signal amount on the calibration surface is independent of the analyte concentration, if the signal amount on the measurement surface is smaller than the signal amount on the calibration surface, it can be said that the analyte is present in an amount greater than the predetermined amount. can. Alternatively, the amount of analyte present in a sample can be quantified by using the ratio of the signal levels of the measurement and calibration surfaces and comparing these to standards prepared using known amounts of analyte. can do. No. 7-16 of U.S. Pat. No. 4,299,916 for a description of various techniques related to the application of the present invention.
See column. This description is incorporated herein by reference. A further embodiment, which is not normally preferred, is to use a combination of enzymes, each binding to the mip and each binding to the measurement surface in proportion to the amount of analyte present in the analysis medium. . The same enzyme combination is then bound to the calibration surface either initially or via binding of the mip complex. At this time, mip can serve as a receptor for each of the two enzymes. Or 2
Enzyme combinations may be linked by using common ligands for the two enzymes. However, in this case, the enzyme molecules may be the same or different enzyme molecules to which the analyte binds. Materials The components used in this analysis are the analyte, the measurement and calibration surface, the signal generation system and, as appropriate, the poly(coordination congener) or polyvalent acceptor. The signal generating system includes at least one catalyst-bound-mip and a solute that is a substrate for the catalyst. Signal generation systems often include additional negatives. Analyte The ligand analyte of the present invention is characterized by being mono- or polyepitopic. Polyepitopic ligand analytes are commonly poly(amino acids), ie, polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Such combinations include bacteria, viruses, chromosomes,
Includes genes, mitochondria, nucleus, cell membrane, etc. The exact nature of the subject and its many examples are
No. 4,299,916 to Litman et al., particularly nos. 16-23.
It is disclosed in the column. This disclosure is incorporated herein by reference. Measurement and Calibration Surfaces The underlying surfaces for immobilizing mips and/or catalysts as measurement and calibration surfaces can vary widely. Generally, the substrate structure is the same for both surfaces and is chosen to minimize interference with the analysis without strongly adhering to the signal generating system. The surface substructures can take different forms and have different compositions, and can be mixtures or laminates of compositions or combinations thereof. The material selected for the surface is capable of interacting with the signal-generating compound by insolubilization of the signal-generating compound or complexation reaction or interaction of other compounds bound to the surface to produce the signal-generating compound; You must destroy or interact with it. The surfaces may take on different types and shapes and may take on different dimensions depending on the use and measurement method. Examples of surfaces may be pads, disks or strips which may be flat, concave or convex. The thickness is not critical and is generally about 0.1-2 mm, but may be of any convenient or other dimensions. Typically, the surfaces are supported on a common member, such as a rod, tube, capillary, fiber, strip, disk, plate, cuvette, etc., but the present invention provides for each surface to be supported on a separate mechanical support. It is also intended to support the top. The surface may form an integral part of the support or be distinct from the support, typically spaced on or from the support and supported by two or more spacers. Apply layers. The surface is typically perforated. Various pore sizes are used depending on the nature of the system. The surface is polyfunctional or can be polyfunctionalized to allow covalent attachment of the mip as well as to allow attachment of other compounds that form part of the signal generating system. The exact nature of the surface is discussed in Litman et al., US Pat. No. 4,299,916, which is incorporated herein by reference. The generation of measurement and calibration surfaces by binding mips to surface materials may be a well-known technique commonly found in the literature. References, e.g. Ichiro Chibata
Author, “Immobilized Enzymes”, Halsted Press,
New York (1978) and Cuatrecases, J.
Biol·hem., 245, 3059 (1970). A wide variety of organic and inorganic polymers, which may be natural and synthetic, and combinations thereof, can be used as materials for the solid surface. Examples of polymers are polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), silicone, polyformamide, cellulose, cellulose acetate,
Contains nitrocellulose, etc. Other materials that may be used include paper, glass, ceramics, metals, nonmetals, semiconductor materials, celmets, silicates, and the like. Furthermore gel-forming substances such as proteins such as gelatin, lipopolysaccharide, silicates, agarose; and polyacrylamides or some aqueous phase-forming polymers such as dextran, polyalkylene glycols (alkylene having 2 to 3 carbon atoms) ) or surfactants such as amphoteric compounds such as phospholipids, long chain (12 to 24 carbon atoms) alkyl ammonium salts, etc. may also be included. Signal Generating System The signal generating system can be non-catalytic or catalytic, in each case amplifying the amount of signal for each mip binding event. In non-catalytic systems, amplification results from having multiple signal-generating functional groups attached to the hub or particle. When a center or particle is attached to a surface through mip bonds, a number of signal-generating functional groups also become attached. Therefore, a constant amplification ratio exists. Common functional substrates used are phosphors and chemiluminescent substances or precursors thereof. Examples of such compounds can be found in the aforementioned US patents. The center or particle may be any simple organic or inorganic core, such as latex particles, glass, etc. These are also well described in the literature. The size of the particles will generally vary from about 10 to 100 microns in diameter. Catalytic signal-generating systems produce a compound that is usually a signal-generating compound, but in some cases reacts with other compounds attached to the surface to produce a signal-generating compound, which can be enhanced or degraded. You may.
Usually at least one enzymatic catalyst will be used in a catalytic signal generation system, although both enzymatic and non-enzymatic catalysts may be used. If only one catalyst is present, this catalyst is conjugated to the mip to bind to the measurement surface by complex formation. In addition to the catalyst, there must be a solute present that undergoes a deformation that results in a detectable signal change at the measurement surface. In many cases, the product resulting from the deformation contacted by the catalyst-bound-mip will be a signal-generating compound. Therefore, if only one catalyst, usually an enzyme, is present, the signal generating system will contain the catalyst-linked mip and its substrate. In some cases it is desirable to attach a "coupler" to the surface, and the catalyst product reacts with the "coupler" compound to produce a signal generating compound. Preferably, a combination of two catalysts, such as an enzymatic and a non-enzymatic catalyst, or two enzymes are used in which the two catalysts are related to each other in that the product of one is the substrate of the other. In this system,
1 undergoing continuous changes catalyzed by a catalyst
Only one solute or substrate is required to produce a compound that is involved in producing a detectable signal. However, in many cases it is usually the first
There will usually be a second compound providing the signal-giving compound that serves as a substrate for the enzyme and a precursor for the compound involved in the production of the signal. That is, the product of the first enzyme reacts with a precursor to the signal-generating compound to provide the signal-generating compound. In most cases the reactions involved are hydrolysis or redox reactions. In the case of hydrolysis, the replacement of the dye with a water-soluble compound linked by an enzymatically labile bond requires two enzymatic steps to give an insoluble dye product. This is an example. In contrast, in the case of a redox reaction, a first enzyme can generate the essential substrate for a second enzyme, where the second enzyme combines the product of the first enzyme with a dye precursor. Contact reaction between. Enzymatic reactions may involve the modification of solutes to products that are substrates for other enzymes or the production of compounds that do not contain a substantial portion of solutes that serve as enzyme substrates. The first situation is exemplified by glucose-6-phosphate, which is catalytically hydrolyzed by alkaline phosphatase to glucose, where glucose is the substrate for glucose oxidase. The second state may be exemplified by glucose oxidized by glucose oxidase, which enzymatically reacts with the signal generating precursor to yield hydrogen peroxide to generate the signal generating agent. Combination catalysts can include enzymes along with non-enzymatic catalysts. Enzymes can produce reactions that undergo reactions catalyzed by non-enzymatic catalysts, or non-enzymatic catalysts can produce substrates (including coenzymes) for enzymes. For example, Meldola blue contains NAD and hydroquinone.
Conversion to NADH can be catalyzed, which reacts with FMN oxidoreductase and bacterial luciferase in the presence of long chain aldehydes to emit light. A wide variety of non-enzymatic catalysts that can be used in the present invention are disclosed in U.S. Pat.
Found in No. 4160645. The relevant portions of this patent are incorporated herein by reference. A non-enzymatic catalyst is a first compound that reacts with a one electron transfer and two
Used as a second compound reactant that reacts by electron transfer. The two reactants can then react with each other only slowly, if at all, in the absence of a catalyst. Combinations of various enzymes can be used to apply signal generating compounds at the surface. In particular, combinations of hydrolases can be used to generate insoluble signal generating agents. Other combinations of hydrolases and oxidoreductases can also provide signal generating compounds. Combinations of oxidoreductases can also be used to generate insoluble signal generating compounds. The following table is an example of various combinations that can be used to preferentially generate signal generating compounds at a surface. As generally discussed above, by appropriate selection of the catalyst at the surface, multiple reagents can be combined in a single formulation. In the following table it is intended that the first enzyme be bound to the surface and the second enzyme be bound to the mip, in particular cases where it is desirable to retain that position.

【table】
It is quite clear that the dyes described above may be substituted with other dyes that have suitable solubility requirements or can be modified to have suitable solubility requirements for the present invention. sell. Furthermore, it should be understood that the highly localized dye concentration causes the dye to rapidly bind to the surface. Furthermore,
The increased amount of dye that diffuses from the bulk solution to the surface will not significantly affect the amount of dye that precipitates on the surface. Depending on the nature of the dye, the absorption of light by the dye or the emission of light, if fluorescence, can be measured. Instead of a chemical reaction to the enzyme product to produce a signal-generating compound, the environment of the enzyme product can be selectively modified upon binding to a surface to produce a signal-generating compound. For example, esters or ethers can be hydrolyzed to produce an insoluble, colorless form of a charge-sensitive dye at the surface. The local charge at the surface can be made substantially different from the bulk solution by having charged groups on the surface. By using signal-generating compounds that are sensitive to the concentration of protons, the signal observed from the surface-bound product is very different from that of the product in bulk solution or liquid phase. Fluorescent quencher pairs may also be used if the solute produces an insoluble quenching product. Supporting Materials Various supporting materials are often used in this analysis. In particular, the analytical medium includes enzyme substrates, cofactors,
Activators, scavengers, inhibitors, etc. may be included. Additionally, buffers as well as stabilizers will usually be present. Frequently, in addition to these additives, further proteins such as albumin or surfactants, especially nonionic surfactants such as polyalkylene glycols, may also be included. Experimental Results The following examples are illustrative and not limiting. All percentages and parts are by weight unless otherwise specified. However, for liquid mixtures, parts by volume shall be used. If no solvent is specified, it is water. All temperatures are Mr. Setsu's unless otherwise specified. The following abbreviations are used:
CMM…O ³ carboxymethylmorphine; HRP…
Horseradish peroxidase; NHS...N-hydroxysuccinimide; EDCI...N-ethyl-N'-
(3-dimethylaminopropyl)carbodiimide; DMF...N,N'-dimethylformamide;
THF...tetrahydrofuran; BSA...calf serum albumin; GO-AMINE...glucose oxidase-amine; Tvifon QS44...anionic surfactant (Rohm and Haas); NaAZ...sodium azide; MOPS...3-N-morpholin Nopropanesulfonic acid (Calbiochem); CDI…
1,1'-carbonyldiimidazole. Example 1 Preparation of Antimorphine Paper The following experiments used antimorphine bound to a paper support made in bulk as follows.
Whatman 1C paper (16 feet x 10.63 inches)
It was wound onto a 1 inch diameter spool using two screens that separated adjacent paper layers and allowed the reagents to penetrate. The resulting cartridge was inserted into a reactor where it was lyophilized under vacuum overnight.
CDI (85 g, Polysciences, lot number 12087, catalog number 15750) was dissolved in 2.5 L of dichloromethane. This solution was recirculated through the reactor for 2 hours using a centrifugal pump at a flow rate of approximately 4 L/min. The paper was then washed three times with dichloromethane (2.5 L) and flushed with nitrogen (approx.
7 L/min) for 3 hours and dried overnight at room temperature. Antibody against morphine (0.2 mg/ml) and GO-
A mixture of AMINE (0.1 mg/ml) in phosphate buffer (2.8 L) was recirculated through the reactor at approximately 2 L/min for 4 hours at 24°C. Then the paper is phosphate buffered (4L)
Wash 4 times with sucrose (15%) and BSA (2
The solution was stabilized with 2.5 L of solution with 10 mg/ml). Remove excess fluid with nitrogen (40psi for 1 minute, 80psi for 15 seconds)
It was removed with . The paper was then subjected to a nitrogen flow (250
ml/min). Example 2 Production of Anti-HRP Paper Two rolls of paper were produced using HRP as follows: A first roll of Whatman 1C paper (12 feet x 10.63 inches) was coated with a pair of wires separating adjacent layers. It was wound onto a 2 inch diameter spool using a screen. A second roll of Whatman 1C paper (16 feet x 10.63 inches) was wound with a 1 inch diameter thread, also with adjacent layers separated using a pair of wire screens. A first roll was placed in a first reactor and a second roll was placed in a second reactor.
Both rolls were then dried under vacuum overnight while flushing with nitrogen to remove moisture. Dissolve CDI (170g) in 5.0L of dichloromethane,
This was recirculated at 4 L/min through the reactors connected in series. The paper was then washed three times with 4.5 L of dichloromethane and then dried with nitrogen (6 L/min) for 3 hours and 200 ml/min overnight. PH the paper from the second reactor with 1.0M NaOH.
Antibody against HRP (29.56 μg/ml) in phosphate buffer adjusted to 6.94, non-immune sheep IgG
(22 μg/ml) and QS44 (2%, w/v). The mixture was kept warm with stirring overnight. GO-AMINE (500 mg) (see below for preparation) was added to this solution along with NaAZ. The final volume was equal to 2.5L. The final concentration of GO-amine was 0.2 mg/min. This solution was recirculated through the second reactor for 5 hours (2 L/min). The reactor was then washed four times with phosphate buffer (4 L) and sucrose (15%).
and stabilized with 2.5 L of BSA (2 mg/ml). Excess liquid is removed under pressure and the paper is placed in a nitrogen stream (2.5L/
Vacuum dried under 1 minute). Glucose oxidase (Sogma, E.
C.1.1.3.4) at a pressure below 30 psi.
It was concentrated from 360 ml to 60 ml using an Amicon PM10 membrane. The glucose oxidase was dialyzed twice against 4 L of water at 4°C and filtered. It was found spectroscopically to have a concentration of 32 mg/ml. Add 51.5ml of this glucose oxidase solution
5.15 ml of 0.2M sodium periodate was added dropwise and the reaction was allowed to occur for 25 minutes. Product, PH4.5
Sephadex G- using 2mM sodium acetate
The main peak of glucose oxidase was collected by 50 2.5×60 cm column chromatography to obtain 91.5 ml of a solution containing aldehyde derivatives. To this solution, in 0.2M sodium carbonate at pH 9.5
6 ml of 3M ethylenediamine was added dropwise and the reaction was allowed to proceed for 3 hours. Then add 10mg/
Approximately 3.9 ml of sodium borohydride were added and the mixture was kept warm overnight and then chromatographed to remove the sodium borohydride. Example 3 Conjugation of Morphine to HRP In a reaction flask, CMM (35.9 mg),
NHS (12.65mg) and EPCI (21.12mg) in DMF (1.1
ml) were put together inside. The mixture was then flushed with nitrogen and stirred overnight at room temperature to produce the activated ester of CMM. Carbonate buffer (PH9.5) 21
To a mixture of HRP oxyamine (1.9 mg/ml) (see below for preparation) in ml of CMM: HRP = 50: activated ester was added in 10 μl increments over 1.5 h while maintaining the reaction mixture at 4 °C. The molar ratio was added to the end point of 1. The reaction mixture was then purified with G50 Sephadex.
0.1M phosphate,
Elute with 0.2M NaCl, PH7.0 buffer and monitor protein. Protein fractions were then collected. 10mg/in 5mM sodium acetate in buffer PH4.5
Add 50 ml of 0.2 M sodium iodate to 5 ml of ml HRP, stir the mixture for 30 minutes, then add G-50
Subjected to Sephadex column chromatography,
Elution was carried out with 2mM sodium acetate buffer at PH4.5. The protein fraction was collected to 29 ml and the mixture was cooled to 4°C and incubated at 4°C in 0.5M carbonate buffer at pH 9.5.
0.2M2,2'-oxy-bis-ethylamine 2.9ml
was added. The PH of this mixture is 1N
Adjust to 9.5 with sodium borohydride-aqueous solution,
The mixture was reacted for 3.5 hours and subjected to column chromatography on Sephadex G-50. The above process was repeated using 400 mg of HRP and 3.5 g of 2,2'-oxy-bis-ethylamine. No significant change in enzyme activity was observed between the original amine and the modified amine with about 4 additional amino groups. Example 4 Preparation of Dip Bars The anti-morphine paper of Example 1 and the anti-HRP paper of Example 2 were punched with 1/4 inch disk holes for use in analysis. The disks were then glued parallel to one end of a plastic rod to form a dip rod. Example 5 Standard curve as a function of concentration of morphine 0.1M phosphate buffer, 0.2M NaCl (2 ml, PH 7.0)
Morphine at 0, 100, 300 and 1000 ng/ml in duplicate was added to the standard solution. Dip rods prepared as in Example 4 were soaked in each solution for 1 minute at room temperature. Without washing, the dipsticks were each placed in 2 ml of developer solution, shaken for 5 seconds, and kept warm for 9 minutes. This developer is MOPS
(50mM, PH6.8), BSA (2mg/ml), 4-Cl-1
- naphthol (0.2 mg/ml) and the HRP-morphine conjugate prepared in Example 3 (20 ng/ml). After incubation for 9 minutes, the dip sticks were removed and the colored paper discs were read on a MacBeth Series 1500 Reflection Spectrophotometer. The results are shown in Table 1. Here RC refers to the calibration disk reflection and RM refers to the morphine disk reflection. RC and
RM is expressed in color difference units given by a MacBeth reflectance spectrophotometer.

The results show that the reflectivity of the calibration disk is independent of the morphine concentration in the test sample, while the reflectance of the morphine disk decreases as the morphine concentration increases. By using the measurement and the ratio of the two values from the calibration disks, a standard curve for the quantification of morphine was created. Example 6 Standard Curve as a Function of Morphine Concentration and Development Time Standard solutions of morphine and phosphate buffer were prepared as in Example 5, except that 8 replicas were made for a total of 40 samples at each concentration. Manufactured. The dip rod was dipped into each of the test samples for 1 minute and then placed in the developer solution (Example 5) for the indicated times. The results are summarized in Table 2.

【table】

[Table] Average
^* average
10 19.4; 20.7
19.9 9.3; 9.3 9.3 0.47
15 24.5; 25.2
25.0 11.9；10.0 10.9 0.44

Claims (1)

[Scope of Claims] 1. In order to determine the presence of an analyte in a sample, where the analyte is a member of a specific binding pair consisting of a ligand and a receptor, one of the labeled specific binding pairs. a member of the specific binding pair, a signal generating system, and a measurement surface bound to one member of the specific binding pair capable of binding to one member of the labeled specific binding pair, with the proviso that the complex of one member of the specific binding pair is the amount of labeled specific binding pair member that binds to the measurement surface as a result of formation is related to the amount of analyte in the analysis medium, the measurement surface and the sample are brought together in an aqueous analysis medium; members of the signal-generating system, including at least one member of the labeled specific binding pair, which simultaneously or successively provide an amount of signal-generating compound at the measurement surface that is related to the amount of analyte in the analysis medium; , bringing together the measurement surfaces, in which at least one member of the signal generating system or one member of a specific binding pair capable of binding at least one member of the signal generating system binds. a calibration surface in the assay medium, the member being capable of binding also to the measurement surface, the calibration surface providing an amount of signal corresponding to a known amount of analyte from the signal-generating compound; , thus the abundance of the analyte is determined by comparing the signal amount at the calibration surface and the signal amount at the measurement surface. 2. The method according to claim 1, wherein one member of the labeled specific binding pair has a central core to which at least one member of the specific binding pair binds to a plurality of chemiluminescent or fluorescent molecules. 3. The method according to claim 2, wherein a plurality of phosphor molecules are bonded to the central core. 4. When an analyte is a member of a specific binding pair consisting of a ligand and a receptor, in order to determine the presence of the analyte in a sample, the enzyme bound to one member of the specific binding pair is binding to a member of a specific binding pair that is bound to a measurement surface using a solute that is catalytically converted by the enzyme that is bound to the measurement surface containing at least one enzyme containing and one member of the specific binding pair; producing a detectable signal change on the measurement surface in relation to the amount of enzyme present in the sample and one of the specific binding pairs on the measurement surface;
an enzyme bound to a member of the specific binding pair relative to the amount of analyte in the sample is bound to the measurement surface by contact with the enzyme bound to the member; having a calibration surface bound in the assay medium in an amount that provides a substantially predetermined ratio to the amount of the enzyme bound to the measurement surface, whereby the amount of signal at the calibration surface versus the amount of enzyme bound to the measurement surface; 2. The method according to claim 1, wherein the amount of the analyte present is determined by comparing the amount of signals on the measurement surface. 5. The method of claim 4, wherein the enzyme is directly bound to the calibration surface. 6. The method of claim 4, wherein the medium binds to the calibration surface during the contact by formation of a complex of one member of a specific binding pair. 7. The signal generating system comprises two enzymes, wherein the product of one enzyme is a substrate for the other enzyme, and one of the enzymes binds to each of the measurement and calibration surfaces prior to said contact. Claims 3, 4,
Any method described in item 5 or 6. 8. The method of claim 7, wherein the solute is a dye precursor that undergoes an enzyme-catalyzed reaction to produce an insoluble dye that binds to the surface. 9. The method of claim 8, wherein the enzyme bound to one member of the specific binding pair is an enzyme bound to an anti-ligand. 10. The method of claim 8, wherein the enzyme bound to one member of the specific binding pair is an enzyme bound to a ligand. 11. In order to determine the presence of an analyte in a sample when the analyte is a member of a specific binding pair consisting of a ligand and a receptor, at least two enzymes comprising an enzyme and a solute dye precursor that is catalytically transformed into an insoluble dye by one of the enzymes binding to a measurement surface comprising one member of a specific binding pair. using a signal generating system with the proviso that the insoluble dye generates a detectable signal change on the measurement surface in relation to the amount of analyte and that one member of the specific binding pair on the measurement surface one member of the specific binding pair through complex formation of one member of the specific binding pair.
providing binding of the member-bound enzyme to the measurement surface;
and contact of the measurement surface with the sample and the enzyme bound to one member of the specific binding pair is related to the amount of analyte in the sample on the measurement surface of the enzyme bound to one member of the specific binding pair. in a method for the analysis of said analyte which results in binding to said measuring surface, during said contacting, a substantially predetermined amount of enzyme bound to said member of said specific binding pair that binds to said measurement surface. of the enzyme bound to one member of the specific binding pair in an amount giving the ratio
a calibration surface adjacent to the measurement surface that binds through the formation of a complex of members, thereby determining the amount of contrast between the amount of signal at the calibration surface and the amount of signal at the measurement surface. The method according to claim 1. 12. The method of claim 11, wherein an anti-enzyme receptor is bound to the calibration surface in a predetermined amount. 13 a second ligand other than the ligand analyte binds to the enzyme of the enzyme bound to one member of the specific binding pair;
12. The method of claim 11, wherein an anti-secondary ligand receptor is also attached to the calibration surface. 14. The method of claim 13, wherein the second ligand is conjugated to an enzyme molecule other than the enzyme bound to one member of the specific binding pair. 15. The method of claim 13, wherein the second ligand is conjugated to the same enzyme molecule as the enzyme bound to one member of the specific binding pair.