JPS58206966A - Simultaneous calibration heterogeneous immunoassay - Google Patents

Abstract

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

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a routine method for quantifying an analyte in a sample suspected of containing the analyte.

There is a continuing 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 mulberry agents, naturally occurring physiological compounds, contaminants, chemicals, impurities, and the like. In the case of fraud, especially in the case of certain physiologically active substances, the speed of measurement! It is important. In other situations, simplicity is the primary consideration.

One simple and quick technique that has found wide application is
It generally comprises a solid rod or film that can be dipped into a sample and subsequently illuminated with a signal that reflects the amount of analyte in the original sample [dip rod (formerly known as a p5tick).
This is the J method. There are many devices available for measuring the signal of a compound bound to a solid surface, such as absorption, reflection or fluorescence.

In addition, the immersion method is easy to handle, move, separate, etc.

Although such techniques are convenient, they have implications for development time, temperature, interfering factors, reagent stability, and observed signal content.
It is very sensitive to other conditions. 12 measured signals! Definitely compared to lI tableware! In the case of li analysis, it is necessary to carefully control the test conditions and adapt the return conditions. However, such "breathing" reduces the convenience of immersion, increases the time and cost 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.

Therefore, a new analytical method, which provides accurate detection of the analyte in a sample and is currently very insensitive to turbidity, interfering factors in the medium, stability of reagents, etc., was developed. It is desirable that the light is open.

Related patents relating to the use of various immobilized reagents and different types of test strips include U.S. Pat. No. 3,993,451;
No. 038485, No. 40465148, l@41294
17M, f#! 4133639M and No. 4160008
@: and includes Gokoku Published Patent No. 2636244. A patent disclosing a method involving separation of bound and unbound antigen is U.S. Patent No. R13,29169, No. 3
No. 949064, No. 3984533,! @39858
67N, No. 4020151, III 403965
2@, No. 4067959, No. 4108972, No. 4
No. 145406 and No. 4168146.

9. At Ippon Komei, wi in a sample suspected of containing an analyte
4i to determine the presence of the analyte! and the method is M
! Served. The method and apparatus include first and second surfaces referred to as measurement and calibration surfaces.

Each preferably contains at least one catalyst and one I
! It includes a signal generation system having 1. Both systems are substantially identical and include at least one common member of the signal generating system.

The measurement surface comprises the binding of a conjugate (C0IIjll(late)) to the surface by means of the generation of a specifically binding pair complex, while the conjugate is a member of a signal generating system that binds to one member of the specific binding pair. (rsps member).

The amount of conjugate bound to the surface is related to the concentration of analyte in the assay medium.

Calibration ostensibly can be covalently or non-covalently - tangentially specific binding! -1 Create the S$IS member that binds to the surface by fF. In this case, the specific binding pair is different from the binding pair of the measurement surface.

By comparing the wrinkles of the signal photobiocompound on each surface, it can be determined whether the edges of the subject are more or less than the predetermined width indicated by the signal generated from the calibration surface.

The present invention provides a method and apparatus for quantifying an analyte in #Xn suspected of containing the analyte by co-introducing at least two different surfaces into a liquid analytical medium. . One surface is then called the measurement surface and the other surface is referred to as the calibration surface. The analysis desirably involves the use of a developmental system having at least one catalyst, usually a substrate, and at least one substrate.

In order to amplify the signal, it is desirable that there be multiple signal silencing events for each binding event (eVellt) of the analyte to its cognate binding device. This plurality may exist as a result of dynamic and static systems. In dynamic systems,
Catalysts, usually used from enzymes, the signal generation system consists of at least one
? 6 substrates, usually containing complementary threads. In this case, U Itomata It
``Any of the substrates may remain bound to the specific binding device. If the substrate cannot be cycled, multiple substrate molecules may be bound to the hub and multiple substrate molecules may be bound to the hub for a single binding event. will provide.

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 more accurately measured with commonly available equipment.
It is used as a signal sealant.

! 1113-membered conjugate and specific binding partner present as a specific binding partner that binds to the measurement surface through liquid association
The amount of IS members is related to the number of subject chicks present in the medium. The number of sps members bound to the calibration surface during analysis will be small and usually one such as to give a favorable range of signals ω for one predetermined analyte depth li. . One sps member on the calibration surface may be present as a result of covalent or non-covalent binding before or during immersion into the analysis solution containing the sample. However, in the case of non-catalytic sps members, the bond is non-covalent and is subsequently immersed into the analysis solution.

The present method and equipment simultaneously calibrate one analytical system during each individual test. 1a @ photoresist silk is associated with 1 on the two surfaces of @ perceivable signals and is subjected to identical conditions with respect to many conditions that influence the observed signals. Therefore, the fluctuations of the detector T8 due to changes in conditions and endogenous substances in the sample will affect the generation of the detector signal on the two surfaces, and the signal drama on the calibration surface will be measured. It can serve as a 41Ii standard for surface No. 16 weight evaluation.

The invention relies on the use of two surfaces with signal light main systems each producing a detectable product. The amount of sps particles bound to the measurement surface is related to the amount of analyte in the medium. The production of detectable products upon sealing off the signal will be directly related to the amount of sps that burns onto the surface. In contrast, the amount of sps member that binds to the calibration surface will not depend solely on the amount of analyte in the medium;
It is usually unrelated to 13-. The set of sps members present on the calibration surface will be independent of the presence of analyte in the analytical medium, even if not pre-bound to the calibration surface.

Once a 5ins-membered molecule is bound to a surface, the raw silk is exposed to the same environment for signals on the two surfaces, and therefore the -I on the calibration surface! The generation of an appreciable signal occurs when the analyte in the medium
It can be used as a basis for qualitative or quantitative analysis of the degree of For many parts, in the case of macroscopic analysis, the calibration surface is not used to determine the specific IA degree, but rather if the intended analyte is opened in more than a predetermined amount. It will be used to determine whether the father exists or not.

When performing an analysis, many measurement procedures and combinations can be used, varying the substances bound to the two surfaces:
! I can do that. It should be understood that in conjunction with two surfaces, 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.

14- Calibration surface includes spa @ binding first to Table @ I @ excluding non-catalytic threads, at which time a predetermined salmon catalyst binds to the surface. In addition, the specific binding pair is 5II
It can be used for calibrating surfaces that bind to S members or for specific binding counterpoints that are exclusive to sps members. Such specific binding pair members are located at the decision point involved in the binding of the analyte to the specific binding pair cognate to the analyte.
t 5ite).

Measuring and calibration surface IJ, any convenient material II to l
It can be constructed in any number of ways, and can have any simple structure. 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 respond 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 [system 15- is at least one functional entity which provides the VA signal;
Preferably at least one catalyst, usually at least one
91, and at least one substrate with sps. In this case, the 5IIS member binds between the specific binding pair to create a conjugate that binds at least to the measurement surface.
It may be attached to both the measurement and calibration surfaces.

The device comprising 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 this case, the two surfaces are introduced into a reagent solution with the necessary substrates and cofactors for the signal-generating system. However, in some situations, e.g. when using two catalysts, For this purpose, the surface of the oscillator system can be combined with the sample in a single analysis medium. After a predetermined time, the surface is removed from the developer solution and the surface to be measured and calibrated are removed. Compare the detected vine signals on the surface.

16- Let us now describe the invention in more detail. However, before describing Ming in detail in the book, we will define Tabe no Jutsu Tsumugi.

l- The analyte may be a monocoordinator, mono- or poly-Epicl<ep+tOpicl, usually the father of the antigen is Ej
A compound or composition to be measured that is an @ body. The compound or compounds share at least one common bitopic point or decision point or receptor.

Specific binding pair ([mip J > - one 7'i of the molecule /
Ji 11! ! Two different molecules with fiRljX on their surface or in their cavities that bind preferentially to the special spatial and polar organization of the 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 diotine-avidin, hormone-hormone receptor, etc. are not immunological pairs. The homologous or complementary substrate is a coordinating receptor;
One-way order l! A purchase is distinguished in a certain state, for example, either a ligator with a collar or an IJ17-Uke.

Coordinator-acceptor: A naturally occurring or manufacturable compound.

Receptor (Anti-coordinator) - A compound or composition that recognizes a special spatial and polar organization of a molecule, ie, an Evitovik point or a decision point. Examples of receptors are naturally occurring receptors such as clopurin, which binds diroxy, and antibodies.

NIFat) fragments, lectins, nucleic acids, etc.

Coordinator homologue - A modified ligand that can compete with a homologue ligand for an acceptor. This modification provides a means for attaching the ligand homolog to other molecules.

Coordination homologues usually differ from the coordination homologs by replacing more than one aqueous system with a bond linking the coordinating homolog center or label, but do not necessarily require a coordinating homologue. What (・.

+1 (coordinator homologues) - An increasing number of ligands or ligand homologs that are covalently bonded together in alignment with the penetrating center.

It is a polyfunctional, usually cliff-coalescent substance having multiple functional groups such as hydroxy, amino, mercapto, and ethylene groups as dead points.

This central core is usually water soluble or at least dispersible and usually has a mass of at least about 35,000 Daltons and generally about 60 Daltons.
It will not exceed 0000 tartons. An example of a central nucleus is
Contains polysaccharides, polypeptides, proteins, IL ion exchange resins, etc.

Surfaces - The measurement and calibration surfaces are each non-dispersive;
Usually present on a common support and as little as about 50μIll
! , generally more than, often at least about 11I11,
In particular, any substance that has a surface area of less than about Q,5ca+2, is insoluble in water, and provides the necessary properties for the binding of a lip and a detectable signal-generating compound to provide the desired amount of signal. good. Desirably, the surface is gelatinous, permeable, hygroscopic, porous, or generally has channels or the like, which may have a substantial void volume compared to the total volume. It will also have a more irregular structure.Depending on the nature of the signal-generating compound to be detected, the surface will be absorbing or non-absorbing, preferably weakly absorbing or non-absorbing. The surface may be transparent or opaque and may be the substance or substances, mixture or product. A wide variety of substances and forms can be used. - properties can be substantially retained, so that substances bound to the C surface remain bound to the surface and do not diffuse into the solution. The structure of both the measurement and calibration surfaces is substantially the same. It is desirable that

Catalyst - Catalyst conjugated to Kaleibakuichi 5ip-11ip, common enzyme. @ medium is a member of the signal generation system, and n+ip is a special engineer 9! ! Depending on the ship order, it is selected to be coupled to the measurement surface.

Kengo sealing system ([sps J) - Signal raw silk is catalytic or non-M media, preferably catalytic, more preferably I
Preferably, two enzymes are used in which the product of one M prefecture is a J1 prefecture of the other; One Kiga,
As something appropriate? 11 enzymes would be included. The signal photosilk emits a detectable signal related to the analyte present in the medium at the measurement surface, while at the calibration surface at least one ligand-receptor pair, e.g. a ligand and a natural receptor. body or antibodies, enzymes and enzymes. The signal-generating compound (J), in whole or in part, binds to the surface and emits a detectable signal (signal-generating compound j emits a detectable signal at the calibration surface; detectable signal at the calibration surface) Regardless of the amount of analyte, the noodles depend on at least one factor.Other substances that may be included in this process include:
When at least two iI complexes are used, they are light for the fluorophore, scavenger for the chemiluminescence reactant and intermediate product.

For simplicity, the member of the signal light silk will be referred to as [sps member 2]. When the term substrate includes coenzymes, sps members are fluorophores, chemiluminophores, M-groups, or! It may be 1 year.

2l-1IA The final analytical medium is prepared for each prior addition of reactants or combinations of reactants and removal from the aqueous medium of the surface and to a different aqueous medium with one or more reactants. may be the result of a prior separation, including the transition of 1j, or a combination of these.

Although there is no need to separate unbound EL-sensitive conjugates from those bound through 1L complexes on one or both surfaces, in many processes, unbound catalysts are A substantially free flux solution will be used. The divine medium involved in the analysis consists of a liquid phase and a homophase that define the measurement and calibration "surface."

For analysis, the surface is placed in an aqueous medium and contacted with a sample, a signal generating system, and an auxiliary substance, either simultaneously or in stages, to generate a detectable signal associated with the surface. The detected signal at the measurement surface is the 41m signal coupled to that surface.
This corresponds to the analyte trout in the sample. Depending on the nature of the signal photosilk and the desired signal detection method, the 22-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.

Other polar solvents, including 111 alcohol, J-terres alcohol, etc., may also contain M-organic organic solvents having 1 to 6 carbon atoms, and usually 1 to 4 carbon atoms. Typically these co-solvents are about 40k
It may be present in an amount of 11% or more, and even less than about 20% total.

The EIH for the media will typically range from 4 to 11, typically from about 5 to 10, preferably from about 6.5 to 9.5. The pH is selected to optimally suppress the signal generating system while still maintaining significant obscuration of specific binding by the receptor. In some cases a compromise will be made between these two considerations. Various buffers can be used to achieve the desired pH and maintain the 11H during analysis. Examples of buffering agents include boroS salts, vA salts, 'carbonates, Tris, barbiturates, and the like. The particular AI cut used is not critical to the present invention, but some mIJ agents may be more suitable than others in a particular analysis.

A reasonable 1 m degree is usually used for carrying out the analysis. A constant iL during the measurement period is generally not necessary, but rapid and large fluctuations are undesirable. The temperature (of analysis) is generally about 10-50°C, more commonly about 15 to 45°C.

The i1 degree of the analyte that can be analyzed is generally about 10→~10-I
s hl, more commonly about 10-6 to 10-13 N-
It will be in 1. Depending on whether the analysis is qualitative, semi-quantitative or quantitative, the detection method used and all the analytes in question will determine the I+negative of the typical reagent.

The concentration of the return reagents depends on the process procedure used, the analyte carrier, the surface-bound m i 1+ and the catalyst-bound 1p.
, varies widely depending on the required analysis f1 degree, etc.

In some cases one or the other of the mips will be used in large excess, and in other steps the sensitivity of the assay, 1, will correspond to the change in the ratio of the mips.

When carrying out the calibrated analysis of the present invention, both the measurement and calibration surfaces need to be contacted simultaneously with the sample and the source 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 accomplished by placing both surfaces on a common support.

However, when carrying out the method of the present invention, it is not necessary to arrange the surfaces on a common support;
It is only necessary that the surface be immersed into the various components of the raw silk at the same time and for the same length of time (otherwise the surface will not have any effect on the performance of the analysis). May be treated independently.

A common support for the surface is conveniently a slat or plastic film, such as those used in conventional immersion methods. 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 divine reagent container. Both surfaces are covered with one of the long dipping rods.
Located at the edge, 25- it is desirable to be able to easily immerse it into a relatively small amount of sample, typically 100 μm to 21. Positioning the surfaces next to each other facilitates visual comparison of the surfaces for making #l final determinations. The surface may be located vertically or horizontally.

As already indicated, more than two surfaces can be used, including multiple measurement surfaces and agitation calibration surfaces, one or both. For example, multiple 111II bodies can be analyzed simultaneously and 2·' or multiple calibration surfaces can be analyzed simultaneously.
Different calibration surfaces may be associated with each of the measurement surfaces for different subjects or may be arranged to provide further uniformity.

Various process procedures using more than one IJ solution may be used. Contacting the solution includes stirring or contacting. It also includes a heat retention step, which generally lasts for about 0.5 minutes to 11M.
Furthermore, it usually changes in about 2 minutes to 30 minutes.

The process procedure will vary with the reliability of the encapsulation system and the nature of the analyte, as well as economic, efficiency, and degree considerations. 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 reagents of the signal generating system are provided separately, all of the reagents can be combined with the sample: or one or more S members can be combined with the sample and then the rest of the signal generating system can be combined with the sample. may be combined with a second solution containing members. For example, Suiken-! Flken-1p can be combined with the sample solution. The substrate and any coenzymes are then distributed between the test It solution and the second solution, referred to as the developer solution, or any remaining SpS members added as a second solution. Alternatively, the sps member may dissolve non-conjugatively and bind to the surface, rapidly dissolving 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 specific binding.

The present method and system can be used for the analysis of any analyte by using a label that produces a detectable signal. Competitive and non-competitive processes can be used; the analyte and the sps-sensing analyte may competitively compete with the congener 111ip on the measurement surface, or they may bind sequentially. . Or? ! If one specimen has multiple binding points, m1 bound to the IJ measurement surface will be lost.
It can serve as a trick between m i 1 of p and 5 ps. By varying the number of molecules on the surface and contained in the sps conjugate, the number of solutions with which the surface comes into contact, and the members of the signal light main system, the desired stability of Pi! Every time,
Process procedures can vary widely depending on user preferences and equipment present. Furthermore, some of the same f4 considerations will depend on the nature of the calibration surface and the manner in which the sps members are bound to the calibration surface 1''.

The generation system may be non-catalytic or catalytic.

A non-catalytic system has a central □ or a sealed image bonded to a particle,
For example, if a plurality of fluorophores or chemiluminescent substances are included, a relatively large signal can be obtained from a single bond. 116! 1p, 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.

Next, this measurement surface is immersed in the sample solution.
Connect to the J connection point and fill this. At this time, the powder-substance conjugate binds to the remaining anti-substance 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 1p. When a substrate is bound to 1p, it is usually located at the center (h
ut>) may be used in multilayers as in non-catalytic systems, with multiple substrates and one or more 1p. ! 1
When one of the 'R's is coenzyme, the coenzyme may be recycled by using two systems. In this case, the result is the production of multiple signal light compounds due to the presence of a single coenzyme.1. These systems are well reported in the literature. When the substrate is not cycled, the substrate will normally be a precursor to the fluorophore or will produce actinic light.

In a catalytic system comprising a single catalyst, usually an enzyme, combined with tm i p, the catalyst-bound-1p and the analyte are.

A competitive sphere format can be used in which the catalyst-bound-1p competes for the homolog 1p simultaneously or sequentially at the measurement surface. That is, a sample containing analyte II was treated with a catalyst.
Binding - May be brought into contact with a surface in combination with Illip;
Alternatively, it can be subsequently contacted with catalyst-bonded-1p'. In the former case, the catalyst-bound-m11) is present in a relatively limited amount and directly competes with the analyte for the 1p binding points present on the measurement surface. In the latter case, catalyst-bond-1
p fills the remaining binding points after binding of the analyte to the measurement surface and is therefore more overweight than in the former situation.
The i11 degree of the subject in question can be present. 1
1) After sufficient time, the surface is bonded to the measurement surface.

30 - Contains cofactors containing compounds attributed to the product that will bind to the surface and give a detectable signal! ! ! May be contacted with prizes.

In other embodiments, a combination of catalysts, especially with at least one enzyme, is used. In this case, one of the catalysts is l! of the other catalyst. Generate living things that are natural. This is a preferred embodiment, particularly in the case of two enzymes, in that it reduces the number of reagent solutions and/or washing requirements and rapidly generates the signal generating compound at the surface.

In this specific example, the measurement surface is not only the mip;
It comprises one of two enzymes, preferably the first enzyme of the system.

WI element-binding-mip is preferably the second enzyme in the system. The first iI'1g product is an essential substrate for the second enzyme, and therefore the other substrates and cofactors required for signal photosilescence are not relevant for the pre-ripening reaction in an aqueous medium. may be combined with a second enzyme. The substrate for the first enzyme is repeatedly activated only when combined with the surface;
The desirability of using a group M solution separated from the mip-containing solution is dramatically reduced. The preferred process steps are:
adding a member of the signal-generating system to the sample; then introducing a surface to the resulting solution; allowing a reaction to occur for a sufficient period of time to produce a detectable change in signal at the surface;
and then comparing the amount of detectable signal on each of the surfaces as a measure of the amount of analyte in the medium.

Another option is to first contact the sample with the analyte and then enzyme-binding-11)! It may be added simultaneously or sequentially with itrade and cofactor. As mentioned above, in step 1, excess enzyme-binding
1p can be used in a competitive situation. At this time, the analyte first binds to its capture m i p on the surface. In particular, different monoclonal antibodies (mO-n
When using oclo++al antibody) on the surface as opposed to enzyme-linked Illip, excess Amounts of enzyme-conjugated-1p may be used. The degree of substrate and cofactor is preferably such that it will be present in such excess that it becomes a limiting factor with respect to the repetition rate of the enzyme; ie, it will exceed the Miberellis 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 has at least one, preferably two binding events, i.e. homolog 1p or enzyme-! 1 will be parallel to the measurement surface in a binding event involving the binding of a coenzyme (containing a coenzyme).

In the case of a non-catalytic signal generation system, 1 connected to the 1p measuring surface
Unlike p, let's bind to the calibration surface. Preferably, the 1p bound to the calibration surface will be of the same type as the 1 bound to the measurement surface, and the two surfaces will have adherents, antigens or receptors 33- as appropriate.

For catalytic No. 18 optical sealing, the calibration surface does not need to contain mips. The most popular catalytic calibration surfaces are surface (covalently or non-covalently bonded catalysts) in which catalytic activity is imparted with a predetermined carrier, i.e. during analytical measurements. The catalyst is bound to the measurement surface and subjected on the calibration surface to the same exogenous factors and conditions as the catalyst, i.e. concentration conditions,
Reagent stability, foreign non-specific and specific interferences, local variations in conditions and composition, etc. are all important factors.
Similarly, the formation of detectable vine products will be affected on both surfaces. Therefore, the main source of variation in the production of signal-generating compounds will be influenced in parallel by the measurement and calibration surfaces.

Parallelism can also be further enhanced by forming the m1 complex on one calibration surface.

This can be achieved in various ways. One method is to provide a receptor for the rlj prime medium that does not significantly affect the activity. llI elementary-bond-m i p3
If 4- is in substantial excess over the amount bound to the measurement surface in the S degree range of the analyte in question, the amount of enzyme bound to the calibration surface will be substantially constant.・However, the binding of HIA to its receptor, especially antibodies, is similar to that of the analyte to its congener 1.
It will be 1.

1p can be further made into 1te by further conjugation with a catalyst-bound analyte.

In the case of a coordination body fluid sample, the desired coordination body can be activated as a 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. If the two ligands are bound to the same catalyst, there will be competition between the measurement and calibration surfaces for catalyst-binding-1p. Catalyst-bond-5
When ip is present in substantial excess, the gate of tactile-binding-1p binding to the calibration surface is not significantly affected by the 11 degrees of analyte. In some cases, the catalyst-
Bond-1p may be present in limited amounts, ie, in no substantial excess over the total bond white present on both the measurement and calibration surfaces. The weight of the catalyst combined with the calibration table will vary with the analyte concentration. Otherwise, the only variation in the amount of catalyst bound to the calibration surface would be as a result of variations in conditions affecting the formation of the 1p complex.

If a non-catalytic sps member or substrate is used, it may have two ligands (an analyte and a calibrator) attached to the center or particle. i.e. the a degree of the conjugate (depending on the Competitive or non-competitive regimes can be used in which the father is present in a substantial excess, where a substantial excess means that the conjugate j > m does not change significantly during the analysis. Taste the room.

Typically, the calibration surface will emit a signal substantially independent of the amount of analyte in the analysis medium. By appropriate selection of the components bound to the calibration surface, a predetermined 111!1
[Detectable tf giving amount! No. 5 will be given. The detectable signal from the measurement surface will be affected 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 constant 1 degree layer of the analyte.

In determining the amount of analyte, the signal amounts of the measured and calibration surfaces can be compared and the contrast used as support for the presence of more than a predetermined amount of analyte. Alternatively, semi-quantitative or window analysis can be performed by subtracting the signal amount at the measurement surface by the signal amount at the calibration surface. In this way, any negative or negative influence on the quality of the analysis medium is virtually eliminated.

If one p in the 5IIS member-binding-1p is a receptor, there is an additional opportunity to place the receptor on the calibration surface that binds to the antigenic point on the acceptor portion of the sps member-binding one receptor. You can have it. If the receptor on the surface is an antibody, an anti(antibody) can be used that may be attached to a convenient decision point on the antibody. In this method, 11J of the S p G member-bond - a + i p acts as an acceptor when binding to the capture 1p bound to the measurement surface and as a high coordinating body when binding to the capture 1p bound to the calibration surface. It can work.

In many cases, the user of the device of the invention will be able to perform as few measurements as possible and as few transfers as possible.
It would be desirable to have Zurukoyoshi in iJ Noh. 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 total testing time.

The simplest process involves applying just enough reagent to the surface.
Alternatively, they may be combined in a suitable formulation, conveniently a lyophilized powder formulation.

If the reagent is delivered to the surface, for example by impregnation, encapsulation, adhesion with aqueous adhesives, etc., it is only necessary that =38-@stomach 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, introduce the surface into the sample solution. For non-catalytic signal generation systems, the surface only needs to be impregnated once by choosing 1p appropriately. In the case of a catalytic system, a single formulation containing two enzymes, one enzyme producing a product that is a component of the other enzyme, can be used. First! If the I element is bound to both the measurement and calibration surfaces, the second S element is bound to both the measurement and calibration surfaces, independent of the pre-ripening reaction! It can be combined with a substrate for the iI cord of I11. The reason is.

The first enzyme is necessary for the second enzyme! This is because no reaction occurs until one compliment is generated.

.;1- (if only one catalyst is used in a three-frame generation system, -.)i of the solution has a substrate for the catalyst and at least 2 Either one solution is used to contact the surface with two solutions separately, or the catalyst-conjugated carrier 111 is solublely bound to the surface and a single substrate-containing reagent solution is used.

The signal generating compound is associated with an increase or decrease in the observed signal.

The signal-generating compound preferentially binds to the surface of the catalyst generated by the catalyst and can be detected visually or by reflectance or fluorescence. is substantially insoluble in the medium in which it is produced and may be derived directly or indirectly from the catalytic ILJ material.

4g I3. If the generated compound is generated on the surface by the surface-bound catalyst, the proportion of total signal generating compound from the solution that results from binding of the No. 4d generated compound to the surface will be minimized.

In many situations, scavengers are desirable. For example, when two enzymes are used in a signal generation system, if a capture agent for the J enzyme is used in the bulk solution, the production of the No. 1d generating compound in the bulk medium can be further reduced. Also, enzymes that do not bind signal-generating compounds in the bulk medium
A capture agent that binds to the signal-generating compound is useful in the bulk medium if the presence of bound iip is to be generated. Enzyme inhibitors may be used that selectively activate enzymes in the solvent, but are substantially inactive to enzymes bound to the surface. This can be achieved by using a barbed inhibitor or a self-destructing inhibitor attached to large porous particles that prohibits access of particle-bound inhibitors to the binding points of surface-bound enzymes. .

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. In the pond
Surfaces colored with certain dyes may be used in which the color observed upon precipitation of different dyes onto the surface 1 changes. There is 1 @1 enzyme in the pond.
Enzyme combinations may be used in which a signal-generating compound is generated and a second enzyme destroys the signal-generating compound or destroys an essential intermediate. For example, when glucose oxidase and horseradish peroxiguase are used on the surface and caralase is bound to mip, the more catalase is bound to the surface, the less dye will be produced.

That is, the third enzyme is present as an enzyme-linked-+++il+, and the enzyme-linked -mil+ is bound to the surface by two.
+ welfare would be associated with reducing the observed production of signal-generating compounds.

For constant m analysis, it is necessary to define the ratio of the signal amount at the measurement surface as related to the signal amount at the calibration surface.

That is, (by setting a specific interval f from the start of the production of the 4-frame generating compound to the end of the further production of the 1.1-frame generating compound, the ratio of No. 1d from the measurement surface and the calibration surface is
The amount of analyte can be related to a standard value for quantifying it. Signal Generation - 42 = If the compound is generated at a constant rate, time is not a critical factor as long as a sufficient change in signal occurs at both the measurement and correction surfaces, but the change in signal occurs no longer at the surface. I do not disguise time so much that it is no longer observed. The yield ratio is relatively insensitive to time, temperature, and endogenous variables. If two correction surfaces are used in the equation, the ratio of the signals on these surfaces can be used instead of measuring time.

The following are examples of two process sequences using a single enzyme and a combination of enzymes. The analyte is a coordinate and the two surfaces have receptors that attach to different coordinates. The second coordination position is then referred to as the calibration coordination position. The enzyme is conjugated to both the analyte cognate and the calibrating ligand. A sample is obtained and dissolved in a predetermined volume of aqueous buffer medium.

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 tear bodies is applied in an amount sufficient to compete with the analyte for receptors 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 left to immerse for a period 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, 2
The two surfaces are separated from the analysis medium and introduced into a developer solution. After a reasonable period of time for the signal-generating compound to form on the surface, the surfaces are removed and the signals on the two surfaces are visually compared. By suitably choosing the amount of +nip in the calibration surface, it is possible to ensure that if the signal from the calibration surface is larger, e.g. That's what I understand. Measuring the signals from the two surfaces,
By determining the ratio, this ratio can then be compared to a standard that indicates the actual amount of analyte present. Semi-quantitative results can therefore be obtained by visual observation, or if quantitative results are desired, by carefully measuring the signals on 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 past sequences, in this case two (enzyme-conjugate combinations) are used, with the same enzyme and two different deposits, 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. The two adhesion bodies should not cross-react, so that the production of each binding complex is similarly sensitive to variations in conditions and endogenous substances.
Preferably, the properties should be similar. The two receptors on the calibration surface are present at a fixed rate that produces a signal-generating compound independent of the amount of analyte in the assay medium. The surface will also have a comparable receptor pool with a first enzyme that produces a product that is a substrate for the enzyme-bound monocoordant enzyme.

The process order is illustrated. The two enzyme-bound monoligands do not compete for acceptors at each measurement and calibration surface. Therefore, the amount of enzyme-bound one calibration ligand bound to the calibration IE surface will not be a function of the amount of analyte in the medium. (enzyme-bound monoligand) will not react with the substituted substrate for the analysis medium in the absence of surface-bound enzyme products, but will require all reagents in a single formulation. One then two samples can be combined together. The enzyme-bound monoligand that competes with the analyte for the mip at the measurement surface-L is determined in relation to the amount of analyte present in the medium. Exist in limited amounts to vary the amount. 46 mentioned above
Similarly, the formulation may contain, in addition to the (enzyme-combined monocoordinator) and the substrate, 171L buffers, stabilizers, excipients, and the like.

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. 1st
The 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 the surface is removed and the amount of signal generated is quantified either by visual observation of the surface or by suitable equipment. do.

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 determined amount. . Alternatively, one can determine the amount of analyte present in the sample by using the ratio of the signal caps of the measurement surface and the calibration surface and comparing these to standards prepared using a known amount of one analyte. The answer lies in quantifying it.

See columns 7-16 of U.S. Pat. No. 4,299,916 for a description of various techniques related to the application of the present invention. This description is incorporated herein by reference.

A further embodiment, which is not normally preferred, is that each mil
The solution is to use a combination of enzymes that each bind to the measurement surface in proportion to the amount of analyte present in the assay medium. The same enzyme combination is then bound to the calibration surface either initially or via binding of the +n i IT complex. At this time, mip can serve as a receptor for each of the two enzymes. Alternatively, the enzyme assembly may be linked by using a common ligand for the two enzymes.

However, in this case the enzyme molecule may be the same or a different enzyme molecule to which the analyte binds.

L Source 1,' The components used in this analysis are the analyte, the measurement and calibration surface, the signal generation system and, as appropriate, the poly(coordination homolog) or polyvalent acceptor. The signal generating system includes at least one catalyst-linked a+ip and a solute that is a substrate for the catalyst. Signal generation systems often include additional negatives.

Lid Examination The coordination body limb specimen of the present invention is characterized in that it is a monoepitopic or polyepitopic specimen. Polyepitopic coordinating body analytes are commonly polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Such combinations include bacteria, viruses, chromosemes, genes, myochondria, nuclei, cell membranes, and the like.

The exact nature of the analyte and many examples thereof can be found in LiLma-n
U.S. Patent No. 4.299.916 of et al., especially No. 16-
It is disclosed in column 23. This disclosure is incorporated herein by reference.

The surface of the substrate on which the MIP and/or catalyst are immobilized and serves as the measurement and calibration surface 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 substrates can take different forms and have different compositions, and may 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 the compound bound to the surface to generate and destroy the signal-generating compound. or have to interact with it.

The surfaces may take various shapes and shapes and may take on various dimensions depending on the use and the particular method. Examples of surfaces may be pads, disks or strips which may be flat, concave or convex. The thickness is not exact, but is generally about 0.1
2, but may be of any convenient size or pond size. Typically, the surfaces are made of common shells, such as rods, tubes, wood,
It is supported by fibers, fibers, IJ tubes, discs, flat plates, cubera) (cuv-50-eLLe), etc., but in the present invention, each surface is separated by a separate Ill? It is also intended to be used as a prescriptive support. The surface may form an integral part of the support or be extended from the support, typically spaced from or to support-L 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 1l
It can be polyfunctionalized to allow covalent attachment of ip as well as attachment of compounds forming part of the signal generating system. The exact nature of the surface is discussed in LiLman et al., US Pat. No. 4,299,916, which is incorporated herein by reference.

The generation of measurement and calibration surfaces by binding of mip to surface materials is a well-known method commonly found in the literature.
? ,)v,. ***,' Mt4r I chir. C
Arrived at hibaLa, “ImmoL+1lized Enz
ya+es'', Ha, 1sted Press.

New York (197B) and Cuatreca
ses%J-[3iol ・Item, 245.30
59 (1970).

A wide variety of organic and black polymers, both natural and synthetic, and combinations thereof, can be used as materials for the solid surface. Examples of polymers include polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(vinyl 1-thylate), rayon, nylon, poly(vinyl 1-thylate), silicone, polyformamide, cellulose, cellulose acetate, Contains nitrocellulose, etc. Other materials that may be used include paper, glass, ceramics, metals, non-metals, semiconductor materials,
Further del-forming substances such as proteins such as gelatin, lipopolysaccharides, silicates, 7-loose;
Mid or some polymers forming the aqueous phase, such as dextran, boria (rukylene glycol (fluorene carbon 1k 2-3)
Alternatively, surfactants such as amphoteric compounds such as phospholipids, fulkylated (12 to 24 carbon atoms) heptammonium salts, etc. may also be included.

No. 1 No. 1 Raw No. 1 The signal generating system can be non-catalytic or catalytic, in each case amplifying the amount of signal for each a+ip 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.

The size of the 9 particles, which are also well described in the literature, will generally vary from about 10 to 100 microns in diameter.

Catalytic signal-generating systems produce compounds that are usually signal-generating compounds, but in some cases 53- react with other compounds bound to the surface to produce signal-generating compounds and enhance performance. Or it may be decomposed.

Usually at least one enzymatic catalyst will be used in a catalytic signal generation system, although both oxygenic and non-enzymatic catalysts may be used. If only one catalyst is present, this catalyst will bind to the measuring surface by complex formation.
Military service to l1ip. There must be a solute present in the catalyst pond 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-IIlip will be a signal-generating compound. Therefore, if only one catalyst, usually an enzyme, is present, the signal generating system will contain the catalyst-binding-mip and its substrate. In some cases the surface [Coupler 1
The catalyst product reacts with a "coupler" compound to produce a signal-generating compound.

Preferably, a combination of two catalysts, such as an enzyme and a non-enzymatic catalyst, or two enzymes are used, where the two catalysts are related to each other in that the product of one is the substrate of the other. In this system, only one solute or substrate is required to undergo continuous changes that are contacted by the catalyst, resulting in one formation of the compound involved in the production of a detectable signal. However, in many cases there will be a second compound which normally provides the signal-giving compound, which normally serves in the system as a substrate for the first enzyme and a precursor for the compound involved in the production of the signal, i.e. The product of the first enzyme reacts with a precursor to a signal-generating compound to provide a signal-generating compound.

In most cases the reactions involved are hydrolysis or redox reactions. In the case of hydrolysis, two enzymatic steps are required to give an insoluble dye product.The replacement of the dye with a water-soluble compound linked by an enzymatically labile linkage is a major problem in this type of system. In contrast to example 6, in the case of a redox reaction, a first enzyme can produce the essential substrate for a second enzyme, where the second enzyme combines the products of the first enzyme. Contact the open reaction with the dye bend body.

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 a substrate for glucose oxidase. The second state may be exemplified by glucose oxidized by glucose oxidase, which reacts enzymatically with the signal-generating compound to yield hydrogen peroxide, producing the signal-generating agent.

Combination catalysts end up including enzymes along with non-enzymatic catalysts. An enzyme can be contacted by a non-enzymatic catalyst to produce a reaction that undergoes a reaction, or a non-enzymatic catalyst can produce a substrate (including coenzymes) for the enzyme. The conversion of hydroquinone to NAD) can be catalyzed, which reacts with FMN oxidoreductase and bacterial luciferase in the presence of long chain aldehydes to emit light.

A variety of non-enzymatic catalysts that can be used in the present invention are described in 1979.
U.S. Patent No. 4.16+1゜6 dated July 1 tl B
Found in No. 45. The relevant portions of this patent are incorporated herein by reference. Non-enzymatic catalysts include a first compound that reacts in one electron path and a second compound that reacts in two electron paths.
used as a compound reactant. The two reactants can then react with each other only slowly, even if this occurs 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. Although combinations of hydrolases and oxidoliguctases can also provide signal-generating compounds, combinations of oxy-Y-ligctases can also be used to generate insoluble signal-generating compounds. are examples of various combinations used to preferentially generate signal generating compounds at the surface. As mentioned above, by appropriate selection of the catalyst at the surface, a large number of reagents can be combined in a single formulation.

In the following table, the first enzyme is intended to be surface bound to the second enzyme; it is intended to be bound to the surface, and in particular cases it is desirable to retain that position.

58- Table 1 Two enzyme systems with interrelationships First enzyme Second enzyme Solute
Signal generating dose 7 toll dye oxidase peroxidase 2 uricase horseradish urate
O-dianisidine dye peroxidase 1 glucose microperoxy β-D-gluco pisutoluison oxidase 4, esterase β-glucuronida 2.2-bis 3',
3"-di(3'-chlor Chlorofuenose + 4/-glutarophthalene'lonyloxyphe'okir) -phthalidocholine chloride nitstyl reaction 1, galactose 10, → D-galactono-δ-lactone + u , 0゜z M, 0.+4-C'1-1-naphthol → dye 1, urate 10, → allantoin 10 M,
u.

2, If, 0. + o-soanisidine → dye 1,
Glucose 10, → D-Dalcono-δ-lactone + M
, 0゜2H heavy O8+bis-toluidine→soft roll 4'-dalcuronyloxyphenyl) -phthalide 22.
2-bis <, 3'1"-chloro-4-gluta = xyphenyl) -phthalide → 3', 3"-dichlorophenolphthalene Table 1 (continued) 11.1□-1--1 -1≠□□-1 word 28111. ．．
--÷--The first # element of life The second element Takashi Yuku
Signal generation 5 Alkaline peroxy 4-C1
-4-C'l-1 phosphatadase l-naphthi-naphtholose ruphosphe dye-to-6 he4-soquina glucose iodonide-rose -6-phos glucose triphenylphate formaza/dehydrogenase 7 alkaline β-galac 0' -(β 4-
Alkylphos 7-atatosidase-D-gala umperiphase tosidyl
(Ron-6'-phosphate) 4-Alkyl9-peripherone reaction 1,4-C"1-1-7terphosnoate →4-C'
1-1-naphthol z 4-C1-1-naphthol-
Dye 1, y, +1/course + ATP → glucose -
6-phosphate 2 glucose-6-phosphate 1NAIJP-! V, 4DPIi Hue 17/Methopurzuate + IWAEH +) Riphenyltetrazolium chloride → Formazan 1, 0" - (β-D-galactosidyl-6'-phosphe-) >4-'Ahakirunheliferon → 01
- (β-D-galactosidyl)4-furkylumpe7
Lifueron 2, 0'-(β-D-galactosidyl) 4-Alkylumenrou/Periferon → 4-Alkylumperifuron It is quite clear that the above-mentioned dyes have suitable solubility requirements or Other dyes may be substituted that can be modified to have suitable solubility requirements for the present invention. Furthermore, due to the highly localized dye concentration,
It should be understood that the dye quickly binds 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 chemical reactions to the enzyme product to produce the signal-generating compound, the environment of the enzyme product can be selectively modified upon binding to the surface to produce the 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. Sensitive to the concentration of protons, the signal observed from the 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.

Torika fishing cup fee Various auxiliary materials are often used in this analysis.

In particular, the assay medium may include enzyme substrates, cofactors, activators, capture agents, inhibitors, and the like.

Additionally, buffers as well as stabilizers will usually be present. Frequently, in addition to these additives, further proteins, such as fulbumin, or surfactants, especially non-ioX surfactants such as polyalcoholene gellicols, may be included.

The following examples are intended to be illustrative and not limiting.

All percentages and parts are by weight unless otherwise specified. However, for liquid mixtures, parts by volume shall be used.6 If the solvent is not specified, it is water. All temperatures are Mr. Serra's unless otherwise noted. The following abbreviations are used: CMM---03 carboxymethylmorphine: HRP---horseradish peroxyguse; N
H8---N-hydroxysuccinimide; EDCI---
-N-ethyl-N'-(3-tumethylaminopropyl)
Carbodiimide; DMF---N,N'-dimethylformamide;THF---Tetrahydro7ran; BSA-
m-calf serum albumin;
E-11 glucose oxidase-amine; Tvif
on QS 44--7 ionic surfactant (Rol
+m and Haas); NaAZ---sodium azide: MOPS---3-N-mol 7 olinoprohanesulfonic acid (Calbioche+n): CD I-
Bow, 1′-carbonyldiimidazole.

Example-1 Trial (/u) Kun 1Cok Preparation The following experiment used antimorphine bound to a paper support prepared in bulk as follows. Wl+atma Ro63-1C paper (16 feet by 10.63 inches) was wound into a 1 inch diameter spool using two screens that separated adjacent layers of filter paper and allowed the reagents to penetrate. The resulting cartridge was inserted into reactor-2, where it was lyophilized under vacuum overnight. CI') 1 (8511, Po1
ysciences, Lotu Y No. 12087, Catalog No. 15750) was dissolved in 2.5 L of cyclomethane. 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
Dry with nitrogen (approximately 7 L/min) for 3 hours and transfer overnight at room temperature.

Antibodies against mono-shin (0.2 mg;/ml) and G
Approximately 2 L of O-AM I-N E (0.1 ms/ml) phosphate buffer (2.8 L) raw stone compound was added at 24°C for 4 hours.
/min through the reactor. The paper was then washed 4 times with phosphate buffer (4 L), souffles (15%) and B
2.5L of solution containing SA (21118/+111)
It was stabilized. Excess fluid was removed with nitrogen (40 psi for 1 minute, 64-80 psi for 15 seconds) and the paper was then vacuum dried under nitrogen flow (250 ml/min) at 89° for 5 hours.

Two rolls of filter paper were made using HRP as follows: WbaLman I C paper (12 feet x 1 l), 6
A first roll of 3 inches) was wound onto a 2 inch diameter spool with a pair of wire screens separating adjacent layers. Wl+aL-810C paper (16 feet x 11.
A second roll of 1.63 inches) was wound with thread, also 1 inch in diameter, 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. and both a
- (under vacuum overnight while flowing nitrogen to remove moisture)
It was dry.

CD I (171 g) in dichloromethane 5, l)
The paper was then washed 3 times with 4.5 L of dichloromethane and then rinsed with nitrogen (6 L/min) 3 times with nitrogen (6 L/min). Dry overnight at 200 s+l/min.

The paper from the second reactor is 1. (1 with 1M NaOH)8
Antibodies directed to IIRI' (29,56 μs, /+l), non-immune sheep 18G (22 μs/+1) and QS44 (2%, w, /+l) in phosphate buffer adjusted to 6.94
v). The mixture was kept warm with stirring overnight. To this solution (io-AMI N to: (
', +00 mg) (see below for manufacturing method) was added along with NaAZ. The final volume was equal to 2.5L. The final concentration of O-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 4 times with phosphate buffer (4 L), sufrose (15%) and BSA (2 kg/
It was stabilized with 2.5 L of feed 1). Excess fluid was removed under pressure and one paper was vacuum dried under nitrogen flow (2.5 L/min).

GIV course oxidase (Sogma, E, C,
1°1.3.4) at a pressure of 30 psi or less
It was concentrated from 360 plow 1 to 60 mol I using a+1conI'M 11) membrane. The glucose oxidase was dialyzed twice against 4 L of water at 4° C. and filtered.

It was found spectroscopically to have a density of 32a+g/ring. This glucose◆oxidase solution 51.
5+1 with 0.2M sodium periodate5. ．． 15m1
was added directly and the reaction was allowed to occur for 25 minutes. The product was purified using 2IIIM sodium acetate for DNA, S.
-pl+adex G-50 2,5X60c+o column chromatography, the main peak of glucose oxidase was collected to obtain 91.5 ml of a solution containing aldehyde derivatives. To this solution was added dropwise 1 part of 3M ethylenediamine in 2M sodium carbonate with 9.5 t) of DH and the reaction was allowed to proceed for 3 hours.

Approximately 3.9+al of sodium borohydride at 10a+B/ml was then added to the mixture and the mixture was incubated overnight and then chromatographed to remove the sodium borohydride.

n example - 67- Reaction of Morch to HRP In 79 sufus, CMM (35,9 g), N
H8 (12,65 mg) and EPCI (21,121)
were combined in DMF (1,1+al). The mixture was then flushed with nitrogen for 7 mL and stirred overnight at room temperature to form CMM.
The activated ester was prepared. Carbonate buffer (pH 9,
5) HRP oxyamine in 21 candy 1 (1,9+++g/
m1) (see below for manufacturing method), while maintaining the reaction mixture at 4° C., the activated ester was added in increasing amounts at 108 m over 1.5 hours until a molar ratio of CMM:IIRP = 50:1 ended. Added. The reaction mixture was then converted to G 505
Apply to a 2 x 30 cm column of ephadex and add 0.
Elution was performed with 1M phosphate, 0.2M NaCl, pH 7.0 buffer and protein was monitored.

Protein fractions were then collected.

10 m in 5mM of lium acetate (pH 4,5 buffer)
0.2 M sodium iodate 501 was added to 5-1 of g,/+1 HRP, the mixture was stirred for 30 minutes, and then G
-50S ephadex column chromatograph 68
- Pour in pl (4,5 2M sodium acetate)
It was flushed out with one buffer. Collect the protein fraction to 29ml,
The mixture was cooled to 4°C and 0.2M 2.2'-oxy-
2.9 ml of bis-ethylamine was added. The r+,H of this mixture was adjusted to 9.5 with added IN sodium borohydride-aqueous solution, and the mixture was reacted for 3.5 hours and subjected to column chromatography on Sel+1+adex G-50.

If RP 41) The above process was repeated using OII+g and 3.5 g of 2,2'-oxy-bis-ethylamine. No significant change in enzymatic activity was observed between the original amine and the modified amine with about 4 additional ami7 groups.

n 泗-”.

The anti-morphine paper of Example 1 and the anti-II l<1' paper of Example 2 were punched with 1/4 inch disc holes for use in non-destructive manufacturing analyses. The disks were then glued parallel to one end of a plastic rod to form a dip rod.

0.1M phosphate buffer, 0.2M NaCl (2+nl
, pH 7, (1) in duplicate) (d
ul mountain C-ate) 0.1 () 0.300 and 100
0 ng/+nI of morphine was added. Dip rods prepared as in Example 4 were soaked in each solution for 1 minute at room temperature. Without cleaning, dip each dip stick into 2 ml of developer solution.
The mixture was shaken for 5 seconds and kept warm for 9 minutes. This developer is MOPS (50mM% pH, 6,8), BSA (
2+ng/ml), 4-CI-1-su7toll (0,2
+g/ml) and the H RP-morphine conjugate prepared in Example 3 (200 ng/ml). After incubating for 9 minutes, emboss the dipstick and place the colored paper disc on the Mac.
Read on a BeLh Series 1500 Reflection Spectrophotometer. The results are shown in Table 1. Here, RC is related to the reflection of the calibration disk, and i (M is related to the reflection of the morphine disk. RC and RM are MacBeLI+ reflection spectra 72-). quantification of morphine by using the ratio of the two values from the measurement and calibration discs. A standard curve has been created for.

Track 1 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. 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.

73- C [morphine] 5 14.1; 11.9
-0 7 14.7i15.7
-10 17.8; 20.7 -15
24.3:24.8 -10
5 14.9H11,7-716,4i16.9
-10 20.1:20.0 -15 2
5.5i25. O-3059,9i11. ? - 716,7i16.9 - 10 21.0720.7 -15 2
4.1i25.4 -100 6
11.2; 10.9 -7 16.9
;16.1 -10 18.1;19.9
-15 24.7i26.3 -3
oo 5 11.6; 11.1
11.8? 14.8; 14.8 1(L
llo 19.4;20.7 19.915
24.5i 25.2 25.0 * Average of all Rc at each time Table 2 Average 9.5i 9.2 9.4 0.791
10;113 1L7 0.781a4;t
s, s 15.5 0.7?21.5:2
α9 21.2 0.847.7r 7.
6 7.6 0.6410, oilO, 7
10,30,64 1&2;15.2 14.2 0.711&
J19.3 1 & 6 α745, li
a8 fLo 0.519.3i
&4 9.4 0.58” i 1 !1
11.0 0.5515, J147
149 0.60m1 5.5 &4
α46a, 2H! L6 Shizuku&4
α466.9i as 6.9 α
439.3 low &3 9.3 0.471
1.9 small α0 10.9 α44! 4
＃-Eshienkaji 16 samples of urine, each from a different supplier, were collected 2...
It was divided into 1 and 1-no parts. Morphine was then added to two portions of each sample to a final concentration of 300 ng/ml. A dip stick (Example 4) was placed in each section and kept warm for 1 hour. The dipstick was then removed and placed in a developer solution (2+++I) prepared as in Example 5. Each dip bar was developed for 9'Iz and the results are shown in Table 3.

75- 76- Disqualification example - Once in the developer solution p↓! -B, 4 animals - 1 Kuhong Yuan body (r41 糺r变杢; upper shadow 1) Two developer solutions were prepared as in Example 5 except that the first solution contained HI<1''-morphine conjugate. Prepared: concentration 150'ng/ml, and second solution 31111
n8/ml.

The results are shown in Table 4.

77 - The results show that the present method and device provide a simple and accurate method for determining the presence of analytes in a medium, both qualitatively and quantitatively. A greatly increased precision of the analysis is obtained by using a calibration surface that is subject to the same effects and fluctuations as the analyte concentration-dependent occurrence of the surface that changes the signal. That is, the presence of a analyte in excess of a predetermined amount can be determined visually, or the results observed by using the ratio of the signal amounts from each surface 77 can be compared to known mouse analytes. 1L with respect to the ratio obtained in the specimen
Graph the changes in the ratio of Mouse No. 1d along with the changes in concentration,
It is possible to make it stationary.

The present method and apparatus offer many advantages for using competitive protein binding assays. Harvesting is simple, quick, and can be done even by relatively unskilled people. Furthermore, the present J method has fewer steps and has a built-in safety factor in that errors other than the measurement of the analyte sample affect the measurement and calibration surfaces alike. Errors are therefore effectively canceled out.

Although the invention has been described in some detail by way of illustration and example for the purpose of providing a clear understanding of the invention, it will be obvious that certain changes and modifications thereof may be practiced within the scope of the claims.

Claims (1)

[Claims] 1. The presence of an analyte in a sample when the analyte is a member of a specific binding body (rlllipJ) consisting of a ligand and a receptor (“anti-coordinator”); using labeled 1p, signal light raw silk and measuring first surface;
However, the amount of MIP bound to the first surface as a result of the formation of MIP is related to the layer of analyte in the analysis medium, and the measurement surface and the sample are brought together in an aqueous analysis medium. and simultaneously or sequentially, at least one member of the signal generating system comprising at least 11p, which provides at the first surface a certain amount of signal generating compound related to the amount of analyte in the analysis medium; ``In the method for analyzing the analyte, comprising the step of bringing together the measurement surfaces, at least one
a calibrating second surface in the assay medium that provides a certain amount of signal from the signal photoreceptor as a result of two ligand-receptor bindings, thereby providing a calibrated second surface at the second surface versus the first The ratio of signals at the surface is substantially independent of non-specific factors.
This method of analyzing an analyte is characterized by limiting the number of analytes in the sample. 2. The method according to claim 1, wherein the #@identified mip forms a central nucleus to which at least one m i l) binds to a greater number of chemiluminescent or fluorophore molecules. 3. The method according to claim 2, wherein an increasing number of fluorophore molecules are bound to the central core. 4. If the analyte is a member of a heterovalent bonding pair + mil + J) consisting of a coordination group and a 6-receptor (U anti-coordinator), the analyte's When determining the presence of a catalyst bound to m i 1+ (1-catalyst one bond-mip
J) and at least one catalyst comprising Il+ i g
Measurement of Containment Using a solute that is catalytically converted by a catalyst bound to a first surface, the amount of signal detected at the first surface is determined in proportion to the amount of catalyst-bound-1p bound to the first surface. a of the analyte in the sample by contacting the first surface with the sample and the catalyst-bound-1p.
1. A method for bonding the catalyst-bound-1p to the first surface in proportion to the W! 41 bonded to the assay medium in an amount that provides a substantially predetermined ratio of the catalyst to the hot water, thereby causing cough!
A method for analyzing an analyte, characterized in that the ratio of the signal at @2 surface to the 3 signal at the first surface limits the amount of analyte in the sample substantially independent of non-specific factors. 5. The method according to claim 4, wherein the catalyst is an enzyme. 6. The method according to claim 4, wherein the catalyst is directly bonded to the M2 surface - □, · " 7. The method during said contact by the formation of a sip complex □ of the l1 body. 8. The photogenic system contains two enzymes as catalysts, provided that the product of one enzyme is the substrate for the other enzyme, and M
8. A method according to any of claims 4.5.6 or 7, wherein one of the prefectures is bonded to each of the first and second surfaces prior to said contacting. 9. The solute is a dye auxiliary that undergoes an M-based catalytic reaction to produce an insoluble dye that binds to the surface.
! #1 The method described in item 4 of the desired range. 10. The method of claim 9, wherein said catalyst-bound-1p is an enzyme-bound monoantagonist. 11. The method according to claim 9, wherein the catalyst-bond-1p is an enzyme-linkage-ligand. 12,, 'The analyte is the ligand and receptor I* ('anti-coordinator J)
When determining the presence in a sample of an analyte when it is a member of a specific binding pair (rmtp J ) consisting of at least two enzymes, including one enzyme bound to 111 is catalytically transformed into an insoluble dye by one of the enzymes bound to the measured 1f! plane containing 1p), with the proviso that the insoluble dye is a detectable signal on the first surface in proportion to the collapse of the object;
p- provides the binding of the enzyme-bound-o+ip to the container surface through formation of a 1p complex, and contact of the sample and the enzyme-bound-11) of the first surface of The W4 of the enzyme-bound-1p is proportional to the amount of analyte.
a method for analyzing said analyte resulting in binding to said first surface, said method comprising: during said contacting, a substantially predetermined ratio to the amount of bound to said first surface; in the amount that gives
The ILj! Ii - has a decoupling second surface adjacent to said first surface to which the binding of 1p binds through the formation of a 1p complex, thereby -)C signal at said second surface versus said first surface;
5. A method for analyzing an analyte, characterized in that the ratio of signals at a surface limits the amount of analyte in the sample substantially independent of non-specific factors. 13. The method of claim 122, wherein anti-US receptors are bound to the second surface in a predetermined order. 14. A second ligand other than the ligand 1s is bound to the S group of the enzyme-bound-11), and an anti-(second ligand) receptor is bound to the second surface. The method according to claim W412. 15. BAW42jii! t&body fermentation group-1 union +n
The method according to claim W414, wherein the method is conjugated to an iD LX extra(7)ll1m molecule. 16. The 21st ii! The vI enzyme-linked n+ i
145. The method of claim 144, wherein p is conjugated to the same enzyme molecule as p. 17, 'Support, measurement first surface of porous material;
A calibrated M2 surface of a porous material near one corner of the surface; a specific binding counter non-diffusively bound to said first surface; and an lIl tree non-diffusively bound to said second surface; 6. Internal calibration diagnostic device, characterized in that it comprises at least one of: t m i p. 18. 1L according to claim 17, in which both the xi and the second surface have the same enzyme non-diffusively bound to the surface; 19; the apparatus according to claim 18; -a+ip 4i: comprising m bound to the surface
i (+ and 1p of the bond -1p are the same but are homologous or both are bonded to different points of attachment of the same -ligand, which are used for diagnostic analysis) Kira for 1-0