JPS62502495A - Direct homogeneity test - 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] Name: Direct homogeneity test field of invention The present invention uses a specific binding substance (specific binding substance). ces). Book In accordance with the invention, the presence or amount of one of the specific binding bare substances can be determined by Complementary binding substances make it possible to determine. especially , the present invention detects whether a specific binding substance is present in a test sample in excess of a predetermined amount. Effective for measuring gargle. The invention also allows a predetermined amount to be added into the test sample. any excess amount of specific binding substance present beyond the . The uses of the present invention are diverse, including the measurement of immunochemically bound substances in biological body fluids, e.g. It is particularly effective for detecting or quantifying antigens or antibodies in human serum.

Conventional technology - Many different evening and eve immunoassays (immunoassays) are currently available. To measure the components of immunochemically bound bears, e.g. antigens and antibodies, in biological body fluids. It is used. Immunoassays generally involve an immunoreactive substance and its immunospecific conjugate ( conjugate or other receptor systems Immunoreactive substances can be measured directly or indirectly by immunoreaction between labeled substances. related to. Even substances that are not immunogenic on their own, such as Habten, is bound to a larger carrier material that may induce antibodies against substances of lower molecular weight. If present, it can be measured by immunoassay. The immunoassay is performed in serum. Useful for detecting immune responses and also for numerous immunochemical methods performed on tissues. used.

The immunoassay is performed using all immunochemical binders in solution or glass tubes or plastic tubes. Immobilized on a solid support such as a stick tube, beads or microtiter plate This can be carried out with epidemiologically reactive substances or immunospecific conjugates thereof. The latter is a solid phase This is known as an immunoassay. Solid-phase immunoassays are easy to perform and labeled Widely used because it is easy to separate the complex from unreacted labeled antigen or antibody. It is being

One type of solid-phase immunoassay includes a direct immunoassay for measuring immunochemically bound substances. There are many non-eompetitive join methods. Measuring antibodies To perform this type of immunoassay, e.g., the antigen is placed on a solid support. fixed and then contacted with a test sample containing the antibody to be measured. Then the sign Another antibody that reacts specifically with the first antibody is added to the test method.

This second antibody is labeled and thus measures the presence of the first antibody in the sample. be able to. This method, commonly known as the "sand f-edge method", allows Foreign antibodies can be easily detected in serum.

Solid-phase immunoassays also target the binding site of an immunospecific conjugate with an immunoreactive agent. Commonly performed as a competitive binding assay based on competition between labeled and unlabeled substances . To determine the amount of antigen present in a test sample using a competitive assay, a test sample is The dye is usually mixed with labeled antigen and then labeled and unlabeled to compete for the binding sites of the antibody. The identified antigen is contacted with the corresponding antibody which is also bound to a solid support. Next, try The test sample is separated into a liquid phase and a solid phase, and the relative amount of labeled antigen present in either phase is determined. Measure quantitatively.

Most competitive binding immunoassays use the same immunoreactive substance present in the test sample. label is attracted to the solid support bearing the immunospecific conjugate with essentially the same affinity. It works on the principle that it can be attached. Therefore, labeled and unlabeled forms of immunoreactive substances will be bound to the support in amounts proportional to the relative amounts of each substance in the sample. for example , the test sample contains 90% unlabeled antigen and 10% labeled antigen, and two If a substance is made to compete for a fixed number of binding sites of the corresponding insoluble antibody, the antibody The ratio of unlabeled to labeled antigen binding is 9:1.

It is also known to perform solid-phase immunoassays using saturation methods. In this case, the fixed The immunospecific conjugate is partially saturated with the immunoreactive substance to be measured in the test sample; Afterwards, the remaining unbound immunospecific conjugate is measured as an amount of immunoreactive material. It binds to the quality label.

Each of the direct competitive saturation methods used to perform a solid-phase immunoassay was used for the assay. Quantitative determination of immunoreactive substances by calculation based on the degree of binding of labeled reagents can be measured.

Radioisotopes, enzymes, and various chromogenic substances are commonly used as labels for the immunoassays. commonly used. Radioisotopes are easily measurable and can directly measure assay results. provides a powerful signal. Immunoassays using such labels generally have excellent assay performance. It is characterized by precision and accuracy.

Enzymes have been proposed as labels for immunochemical binders, especially in home assays. There is. A notable advantage of enzyme labeling is that enzyme activity is not as expensive as macroscopic or colorimetric measurements. This means that it can be detected by a detection device. Immunoassay using enzyme-labeled reagents To carry out the determination, the enzyme activity is determined by the enzyme-catalyzed reaction (typically the light absorption is qualitative). or the formation or disappearance of colored compounds that can be easily measured either quantitatively) measured using an appropriate substrate that provides Immunoassays using enzyme labels are commonly used. It is commonly called enzyme-linked immunosorbent assay (ELISA).

EL I SA method using the so-called "test strip" or "immersion stain" method It is known in the prior art to carry out.

These assays use water-absorbing substrates or carriers. These are natural or synthetic polymers - typically a cellulosic material such as filter paper; The immunospecific conjugate of the immunoreactive substance determined is immobilized. Enzyme label supports an immunospecific conjugate bound to or one member of an immunochemical binding pair in a test sample. can be matched. These assays are usually based on standard EL I SA methods. It is intended to eliminate operational steps such as water washing and constant temperature maintenance. do this This reduces the chance of errors and allows for use by untrained personnel. . Furthermore, this method can be performed quickly and gives macroscopic results. , these assays are preferred for use in the clinic or home.

An ELISA assay using antibody-conjugated test strips is described in U.S. Patent No. 4. No. 168.146.

This test is based on the "Sandinch method" described above. When used, has antibodies Dip the test strip into the test sample suspected of containing the antigen to be measured. Ru. The test sample moves along the test strip until all the antigens present in the test sample are absorbed. After reacting with antibodies bound to the test strip, the test strip is labeled with an enzyme label. Contact with a solution containing bound antibody and enzyme activity measured as described above. .

Another test strip type assay using a combination of enzymes is described in U.S. Pat. , No. 435,504.

The test strip consists of a porous support capable of solvent transfer, multiple specific binding pairs of substances (e.g. antibodies) and enzymes; the latter two The components are bound non-diffusively and uniformly to the support to define the immunoadsorption area. ing. Additionally, enzyme-labeled specific binding pair elements (e.g. (e.g. enzyme-labeled antigen), which is used to bind the target substance (e.g. antigen) to be measured. The portion of the immunoadsorbent region that binds to the immunoadsorbent region and is present in the test sample. Establish clear boundaries related to the amount of target substance to be used. The enzyme label is preferably an immunolabel. Related to romatograph enzymes and discloses one substrate as the product of the other has been done. In practice, immunochromatography is performed in such a way that the test sample moves through the immunoadsorbent region. contact with the test sample for a sufficient time to allow movement. Immunochromatography also uses solvent contact with a labeled specific binding bare substance in a medium, resulting in a labeled specific binding The bare substance binds at the immunoadsorption area with respect to the target substance bound to that area. after that , the immunoadsorbent region contains a substrate that produces a measurable signal upon enzymatic labeling. contact with the developing solution and quantitate the amount of target substance in the test sample. Measure the distance of the border from one end of the romatograph.

Another assay using test strips with enzyme-labeled immunochemical binding substances An example of this can be found in US Pat. No. 4,446.232. When used for antigen measurement The test strip contains an antigen and an enzyme-labeled antibody capable of immunochemically reacting with the antigen. A color-forming reaction occurs between the first region containing the body and the enzyme label bound to the antibody. a second region containing the substance to be obtained, thereby controlling the presence of the antibody in the second region. presence is shown. Antibodies are diffusely bound in the first region; The test sample passes from the first region to the second region while reacting with the moving antigen. However, in the absence of mobile antigen, it will not diffuse through the first region. in implementation The test strip knob should be adjusted sufficiently to allow the test sample to move along the test strip. contact with the test sample suspected of containing the target antigen for an extended period of time and detect the presence or absence of a color change. The absence is observed as an indicator of the presence or absence of the target antigen in the test sample. this The assay can include multiple separate regions, each containing a different amount of immobilized control antigen. It is disclosed that the method can be applied to quantitative measurements.

The location of the color change then depends on the concentration of antigen in the test sample.

The test strips proposed so far for carrying out ELISA methods are immunochemical Less complex and comparative method for qualitatively and/or quantitatively determining binding substances Although rapid methods have been provided, these prior art assays require Quantitative measurements of target substances or any excess amounts of target substances beyond the predetermined amount are not permitted. stomach.

Main place Higashi chestnut carp The invention determines the presence of a binding substance in excess of a predetermined amount in a test sample and To provide a simple and convenient method and test kit for quantifying such an excess amount. Book In order to carry out the method of the invention, it is necessary to use a material that can bind to the binding substance and that already has a binding substance. An array of complementary binding agents having a defined binding capacity is provided. Complementary to test sample An array of binding substances is provided.

Contact with the binding substance or complementary binding substance in the test sample for a sufficient time to bind . A carrier medium containing a fixed amount of labeled binding substance is then added to the array of complementary binding substances. contact with A. A fixed amount of labeled binding substance is added to a predetermined amount of binding substance to be measured. When added, the amount is such that the binding capacity of the complementary binding substance is sufficiently high. then , measuring the absence or significant presence of unbound labeled binding substance to determine if the binding substance is already present. Identify whether or not it is present in the test sample in excess of quantification. Complementary binding of substances The combined volume is complementary to the predetermined amount of binding substance to be measured (if present in the test sample). The total amount of labeled binding substance that is contacted with the array of binding substances is substantially The significant presence of unbound labeled binding substance indicates that Indicates that the binding substance was present in the solution in excess of a predetermined amount. unbound label The amount of binding substance is also measured quantitatively to determine how much of the binding substance in the test sample exceeds a predetermined amount. It is possible to measure whether the level is exceeded. The array of the present invention has a bound/free (B/F) characteristic. Homogeneous in that it does not require separation of differentially binding pair substances called an array.

The test kit of the invention consists of the materials and equipment necessary to carry out the method.

This test kit includes: 1) an array of complementary binding substances on a support; 2) have a predetermined binding capacity for the binding substance to be used; and 2) in addition to the predetermined amount. sufficient label binding to substantially fill the binding capacity of the complementary binding substance when Contains sexual substances.

The methods and test kits of the invention address chemical imbalances in various biological and other fluids. Provide an easy, fast and reliable way to measure things. The present invention is medical It can be used particularly advantageously for diagnosis and detection of relatively low-grade drugs in body fluids. .

Kaien Iυl Minggata-Hari The present invention provides a specific binding bare substance consisting of a binding substance and its complementary binding substance. Concerning the qualitative and quantitative measurement of two substances. In the following description of the invention, in fact Whether any of the bound bare substances is measured using the methods and test kits of the invention. However, the bound bare substance to be measured is referred to as a binding substance.

The term "binding substance" refers to the binding of two substances in mutually specific amounts. Any substance that reacts with a complementary binding substance based on binding affinity. The term “combining "Substances" as used herein include proteins, hormones, polypeptides and Including, but not limited to, both steroids, carbohydrates and glycoproteins do not have. Typical examples of specific binding bears include antigens and their antibodies, haptens and their antibodies. , hormones and their receptors, vitamins and their receptors, and toxins and their receptors There is.

All these measurements are within the scope of the present invention.

The term "complementary binding substance" refers to the specific binding partner of the binding substance being measured. Main departure Complementary binding agents used in the practice of testing are generally not derivatized; This means that the complementary binding substance is not bound to any other chemical moiety or It means not conjugated. Complementary binding substances are suitable provided in the form of an array on a suitable support. The support is glass or plastic. Forms such as capillary tubes, strips of paper, polymer beads, columns packed with suitable substrates, etc. is possible. Those skilled in the art can immobilize complementary binding substances on such supports. There are many well known methods. Such methods include, for example, covalent bonds as well as Other types of fixation are used, such as absorption. Avoid significant diffusion of bound substances Any method can be used. The complementary binding substance is preferably a one-dimensional lattice. is applied to the support so that binding and saturation of the target substance begins at one end and ends at the other end. This means arranging the binding sites linearly so that they proceed uniformly. This is long The binding substance is arranged on a three-dimensional support that is significantly larger than the width, height, or cross-sectional area. This is achieved by Or, the connected component is a two-dimensional support whose length is significantly greater than its width. Can be fixed to the carrier.

The "predetermined amount" used as a standard in practicing the present invention is specific to the binding substance in question. can vary depending on the type of test and the nature of the assay being performed. For example, raw In the case of body fluids, the amount is considered to be the normal or average amount, commonly referred to as the "legal standard amount." There are established values for the various components that can be obtained. The present invention can be used as an immunoassay. The predetermined amount of binding substance to be measured is generally a clinically standard amount.

The following table illustrates clinical standard amounts for some of the various components of human serum.

σ-r antitrypsin 210-510 mg/di ceruloplasmin 20-3 5 mg/di cholesterol 150-280 mg/dl creatinine 0 ．． 8-1.2 mg/difolate 5-21 ng/ml Gastrin 30-200 pg/ml IgG 720-1500 mg/dl IgA 90-325 mg/di r　gM　45～150　mg/di Lipid 450-100 mg/di Albumin protein 4.0-5. 0 g/di globulin protein 2.0-3 ゜Og/diquinidine 4-6 mcg/ml Serotonin 0. 1~0. 32 mcg/ml thyroxine (total) 4-11 mcg/dl thyroxine (free 0.8-2.4 ng/di uric acid 3. 0-7. 0mg/100ml1 Vitamin A 0. 15-0. 6 mcg/ ml vitamin B1z 330-1025 pg/ml complementary binding substance array In addition, the method of the invention includes a method for measuring binding substances or complementary binding substances to be measured. This includes the use of labeled substances that have the same binding affinity as the binding substance being determined. . Suitable labels include radioisotopes, enzymes and chromogenic substances. chromogenic substance U and V of the electromagnetic spectrum.

Or=? There are dyes and fluorescent or phosphorescent materials that absorb visible area light. The above mentioned Suitable methods for attaching any of the above labels to the type of binding substance are well within the skill of those skilled in the art. well known. An important requirement in methods for labeling binding substances is the identification of a specific binding bear. Does not structurally interfere with the reaction site of either component or is not to interfere. Radioactive or phosphorescent labels are Little interference with stereochemical properties.

The target-binding substance is an antigen or a hubten, and the target-binding substance is an antibody to the antigen or hubten. In systems with arrays of complementary binding substances, the labeled binding substances are covalently bound to the enzyme. can consist of combined antigens. Of course, in this embodiment, the antigen for the antibody or antigens or halves in such a way that the affinity of Habten is not impaired or reduced. The enzyme must be attached to the butene.

Enzymes that later show enzymatic activity in the conjugated form include catalase, peroxidase, and lucuronidase, glucosidase, galactosidase, urease as well as e.g. oxidoreducts such as glucose oxidase and galactose oxidase There is Tase.

An important feature of the invention is that the predetermined amount of binding substance to be measured (i.e. in the case of an immunoassay) clinically standard amount), labeled amount and available binding sites of complementary binding material on the array. This means that there is a known relationship between the values of the orders. The binding substance and label are the same. Substantially the same affinity for one substance or complementary binding substances on an array Any binding substance present in the test sample is a complementary binding substance. Competes equally with the labeled binding substance for available binding sites on the substance. complementary conclusion The array of compound substances is tested for binding substances using clinically standard amounts of binding substances and this method. A known has a binding capacity of Presence can be easily measured. (and the amount of the binding substance exceeds the clinical standard) Contains all unlabeled and labeled binding substances, if present in the test sample. There are not enough binding sites on the array for A surplus or spillover is produced. Unlabeled and labeled binding substances Complementary binding substances compete with equal affinity for available binding sites on the array. The effluent therefore ultimately always contains label-binding substance. As mentioned above, Relative amounts of each of the unlabeled and labeled binding substances in contact with complementary binding substances Al/I This is because they combine in proportion to. Therefore, there is no unbound labeled binding material in the effluent. Significant presence indicates that the binding substance in the test sample exceeds an established standard amount. It is recognized whether it is present or not. As used herein, the expression "significant" ``Presence'' refers to an amount that is two to three times the bank background noise or error value in the test system. cormorant.

During implementation, test samples that are thought to contain the binding substance to be measured should be , complementary binding for a period of time sufficient to cause binding between the complementary binding substance and the binding substance Contact with an array of substances. Available binding sites for complementary binding substances are found in the test sample. is filled up to the amount of binding substance present in the The array is then labeled in the carrier medium. Contact with a binding substance. Suitable carrier media include aqueous solvents or biological systems, e.g. include other polar solvents such as ethers or alcohols. Non-polar solvents are It can be used in other systems if appropriate. The carrier medium is slowly adjusted to the desired pH range. detergents can be added to weaken non-specific binding. . Labeled binding substance, if the test sample contains a clinical standard amount of binding substance. substantially fills the remaining available binding sites for complementary binding substances on the array. I think that the.

As an alternative to the sequential method described, the labeled binding substance may be added simultaneously with the binding substance being measured. can be placed in a test sample for contact with an array of complementary binding substances.

According to this method, labeled and unlabeled binding substances are placed in the available binding sites of the array. compete directly against In this method, the test sample is used as a carrier medium.

To carry out the method of the invention for measuring specific antigens in human serum samples, The number of sites provided in the array of combined antibodies is the same as the clinically standard amount and when performing the present method. The amount of antigen corresponds to the amount of labeled antigen that is added. available on array The number of possible binding sites is known, and the clinical standard amount of Hn for the antigen in question is known. so that the amount of labeled binding material used will substantially fill the array as desired. can be changed so that For example, the clinical mark *ffi for an antigen is If filling the 60 binding sites of the body array, there are e.g. 120 available binding sites. The array has an amount of labeled antigen that substantially fills the remaining 60 binding sites. Oranges are also served and used together. Of course, it is desirable to vary the number of binding sites. In some cases, it may even be necessary to change the amount of label used just to achieve the intended result. That's fine. The array was contacted with both the test sample and the labeled antigen (as described above). (either sequentially or simultaneously), 60 or more units of antigen are present in the test sample. If so, not all of the labeled antigen will bind to the array. Test after contact with array Is there no residual label present or significant presence in the sample (or carrier medium)? determine whether the specific antigen in question is present in serum in excess of clinically standard amounts. is shown.

The method of the invention can be used in the clinic or at home in the form of a simple test kit. Test kick The plate contains a phase with a predetermined number of binding sites along with a predetermined amount of labeled binding substance. A suitable support having an array of complementary binding substances immobilized thereon is included. Label binding substance The amount of is dependent on the clinical standard amount of substance being tested and the number of sites on the array. child When enzymes are used as labels in test kits such as No special detection equipment is required. This kind of test is saliva can be performed by relatively untrained personnel with body fluids such as urine. Let's come.

In accordance with the present invention, the amount of binding substance in a test sample exceeds an established standard amount is quantified. It is also possible to measure the Serum containing specific antigens in excess of clinical standard amounts When performing the above method on a test sample, the test sample or carrier medium is The effluent obtained after incubation (eff!, uent) contains unbound label and Contains unlabeled antigen. The amount of unbound labeled antigen in the effluent is then measured. I can do it. In the case of enzyme labeling, the enzyme activity of the effluent can be determined by e.g. color intensity using a colorimeter. It can be measured by measuring. If radiolabels are used, Radioactivity can be measured. Amount of labeled antigen in the effluent test solution or carrier medium When measuring the binding of labeled and unlabeled antigen, the binding of labeled and unlabeled antigen is The amount of antigen initially present in the test solution is easily calculated. I can calculate it. Specific measurements using standard curves as well known in the art It can be performed.

For example, using a column containing an array of antibodies with 300 binding sites, 220 units if the clinical standard amount of antigen in the test sample occupies 80 binding sites. Add labeled antigen to the test solution. If more than a clinically standard amount of antigen is present in the test sample. Whenever this occurs, there will be a corresponding increase in the amount of labeled antigen remaining in the effluent test solution. can be detected. The theoretical amount of labeled antigen is shown in Table 1 below. This amount is 22 0 units of labeled antigen was mixed with increasing amounts of unlabeled test antigen, and 300 When performed on an array of antibodies with binding site capacity "spilled" into the effluent It is something.

X to overflowg 1-00 20 13.8 120 40 25.8 140 60 36.6 200 120 62.9 500 420 12B Efflux plotted against test antigen (>80 units) present in test solution Plotting the activity (labeled antigen in “spill-over”) yields a curve that is essentially a straight line. It will be done. Once this characteristic table is created for a certain antigen and its antibodies, In order to find the amount of antigen present in a test sample in excess of the standard amount of 9p, The enzymatic activity of the labeled antigen in the liquid may be measured.

In another embodiment of the invention, the array of complementary binding substances is It can be immobilized on a solid support with an indicator substance on the solid support. for example It is usually desirable to test serum samples for antigenic components present in amounts of 80 units. What you have will be provided. 220 units of enzyme-labeled antigen are then added to the test sample or separately. of carrier medium. Another area of the same solid support adjacent to the 300 site array In this method, an indicator substance such as a substrate that changes color under the influence of an enzyme label is immobilized. It's okay to stay. There are then 300 bonds available before reaching and contacting the indicator substance. A test solution is brought into contact with the array to fill the site. Antigen present in test solution amount greater than the clinically standard amount of 80 units (if not, both labeled and unlabeled parenchymal Generally, all antigens bind to the array of antibodies, and all antigens reach the indicator region of the support. Not reached. However, if the test sample is greater than the clinically standard amount of antigen, 80 units. , the labeled antigen reaches the indicator substance on the support and becomes visible to the naked eye due to a color change. A signal is generated that can be done.

The latter embodiment of the invention allows the binding substance in the test sample to exceed clinically standard amounts. It is possible to quantify even the amount that is present.

Regarding the above example of using an indicator substance, a solid support that reacts with the label of the immunoreactive substance The indicator substance region of a carrier is related to the amount of substance present in excess of an established standard amount. Ru. The area of indicator substance that reacts with the label is the data reported in Table 7 for excess amounts. and have essentially the same functional relationship.

be written so that the test sample can easily diffuse along the support by capillary action. The solid support in the embodiment described above may be a porous cellulosic material, such as a long strip. It is preferable to use wrap paper. Indicator substances such as immunoreactive substances and enzymes can be directly bonded to a paper support by methods well known in the art. Wear.

The following examples describe methods and procedures for making and using the invention and put into practice the invention. This description describes the best mode presently contemplated for the invention, but does not limit the invention. It should not be interpreted as such.

Disaster - Ship - 91111 This example demonstrates how DNP and DNP-derivatization were performed in accordance with the present invention. d) Describe the manufacture of test equipment used for protein measurements.

Rabbit antiserum against DNP contained 40% glutamate, 30% lysine and 3 Any dinitrophenylated chain polypeptide consisting of 0% alanine It was produced by immunizing rabbits. In this synthetic polypeptide, 10% of the lysine is DNP. It was connected with. D.N.P. Bovine serum albumin (DNP+.

Measuring the titer of the antiserum for DNP using precipitated kidney assay with BSA) The amount of antibody was 2.1 mg/ml of serum. Antiserum is sodium carbonate/Sepharose /Cuffed with Sepharose 4B by cyanogen bromide activation method. Sepharo The antiserum was prepared by adsorption with rabbit BSA anti-BSA precipitate before camping with was decomplemented, dialyzed against a buffer (0.1 M NaHCOs), and microcaraminated. centrifuged in a vacuum chamber. To achieve uniform coupling, all reagents were added rapidly. Mixing resulted in an adsorbent slurry.

The microcolumn containing the adsorbent slurry has a diameter of 20 mm as the body of the microcolumn. It was made using a disposable micropipette from Little. 10-15cm length In order to use a plastic tube with an inner diameter of 1.5 mm as an outflow tube, a micropipette was used. Attached to the bottom of the tray. Before attaching the plastic tube to the end of the micropipette, A piece of cotton or fabric was inserted into the end of the plastic tube. on the end of the micropipette When installed, the fabric or cotton material hits the bottom of the micropipette and absorbs the sorbent. formed an impenetrable barrier. To facilitate the packing of microcolumns For micropipettes, use standard laboratory plastic pipette tips. Take the funnel made by slicing the end of the tube to allow insertion into the micropipette. I attached it.

The microcolumn was first filled with phosphate buffered saline containing 1% bovine blood albumin. water (CPBS) to reduce non-specific adsorption for sub-nanogram values. The mixture was kept warm for 1 hour. The column was suitably packed and filled with the adsorbent slurry. Each force The ram reduces the bed volume to 2 by removing excess adsorbent using a needle or syringe. 0 microliter. The column is 69-72 mm high and 0.5 mm Filled consistently within the range. As a result, the bed volume was controlled within 0.7%.

The microcolumn was measured using radioactive DNP, BSA. 280 and 3 DNP+, aBSA carefully determined by appropriate absorption measurements at 60 nanometers was labeled with iodine-125 by the iodine method. 6 micro curie/micro gram activity and no binding of reasonable amounts of labeled protein when diluted with unlabeled protein. Therefore, since it was combined with a mini column (100 to 300 microliters), It was found that the labeled protein was not impaired by labeling.

To measure the microcolumn capacity, the saturation value of the labeled antigen was measured on seven microcolumns. It was placed in each room. The labeled antigen was placed in a microcolumn at 20 to 80 microlift I. −Given in volume of 1.

After loading the labeled antigen into the column, add 5 times the volume of 1% BSA/PBS to the column. I let the ram pass. The reliability of the number to the combined average Callan 1 is introduced into Callan J, The deviation was approximately 5% regardless of the number of counts. Table H describes the capacity.

1 6.500,000 570,4602 10, OOO, OOo 610. 3103 8, 500.000 585.7004 2.000,000 62 0,1205 1,200,000 605.3606 800.000 578.200 7 650, 000 592.450 The average combined count was 595,660. From the specific activity of diluted antigen , which corresponds to 440 nanograms of antigen. Describes an experiment in which labeled DNP/BSA was added to the test sample to simultaneously provide BSA. do.

A 460 nanogram capacity mini-column was made as described in Example 1. Already A sample containing a known amount of unlabeled DNP/BSA was prepared at 200 nanograms (260 nanograms). 000 counts) of standard mDNP/BSA and applied to a microcolumn.

After applying the test sample, add 7 times the column volume of BSS A/P BS to each column. The effluent was collected in a counting vial and counted. Table shows the results of two trials for each sample. Shown in ■.

From the results of these experiments, the bank ground counts are 0°240 and 260 nanometers. The collected value is collected in grams, while the collected value is set to bank ground when the column capacity is exceeded. It can be seen that the concentration increases rapidly (starting at 280 ng).

Dai-Koichiyuu-1 The experiment of Example 2 was repeated by adding labeled and unlabeled antigens DNP/BSA to the test device at the same time. Rather, it was given continuously.

The test sample was first applied to the column at a volume of 30 microliters, followed by a 20 microliter volume. Chloritol buffer, then 200 nanograms (260,000 counts per minute) ) labeled antigen was given.

The results of this experiment are shown in Table ■.

This experimental result shows that the radioactivity of the effluent increases substantially, so this method is not recommended. It has been shown that this can increase anger levels.

This is due to the fact that no initial dilution with unlabeled antigen has occurred. In two sample cases Since more counts are present in the effluent than were introduced, a small error exists. existence is recognized. However, this error is considered experimentally acceptable.

Disaster M↓ This example demonstrates another method that has been found to be effective in measuring lower levels of antigen. An experiment using the measurement system will be described. For this experiment, reduce the antibody level on the adsorbent. Therefore, the rabbit antiserum was first diluted 1:5 with normal rabbit antiserum. microphone The 20 microliter column was measured as described above. It was shown to have a total capacity of antigen of 120 nanograms. When performing this experiment, 60 nanograms of labeled DNP/BSA (80,000 counts per minute) was and passed through the column. Collect the effluent and count as described above. did. The results of this experiment are shown in Table V.

Table■ The methods and test kits of the invention are described with particular reference to their use as immunoassays. However, the invention has other uses as well. For example, the invention may be Specific binding bare substances present as harmful contaminants in groundwater exceeding established standards (e.g. DDT and naturally occurring DDT-binding substances). Ru. Thus, the methods and test kits of the present invention can be used to test groundwater in accordance with the requirements of federal regulations. When used in analysis, predetermined quantities are criteria established by relevant rules.

The methods and test kits of the invention are described herein in terms of certain preferred embodiments. However, various other embodiments will be apparent to those skilled in the art. Therefore, the present invention actually without being limited to the embodiments described herein, and departing from the spirit and scope of the appended claims. (Changes and modifications are possible.)

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Claims (25)

[Claims] 1. A pair consisting of a binding substance and a complementary binding substance that have mutually specific binding affinities method of determining whether one of the substances is present in a test sample in excess of a predetermined amount The method: a) using an array of complementary binding substances having a predetermined binding capacity for said binding substance; meaning; b) said array of complementary binding substances: i) any binding substance present in said test sample binds to said complementary binding substance; contact with said test sample for a sufficient period of time to ii) contacting a labeled binding substance in a carrier medium, wherein the amount of said labeled binding substance is sufficient to substantially fill the binding capacity of said complementary binding substance when added to a predetermined amount. the array of complementary binding substances and the labeled binding substance are contacting the complementary binding substance for a sufficient period of time to bind the complementary binding substance; and c) determining whether said binding substance is present in said test sample in excess of a predetermined amount; determining the absence or significant presence of unbound labeled binding substance to separate; A method consisting of. 2. Claim 1, wherein the carrier medium for the label-binding substance is the test sample. The method described in Section 1. 3. The test sample is human serum, the binding substance is an antigen, and the complementary Claim 1 or 2, wherein the binding substance is an antibody specific to the antigen. Method. 4. Claim 3, wherein the predetermined amount of antigen is a clinical standard amount of a volume of human serum. The method described in section. 5. 4. The method of claim 3, wherein said antibody is arrayed on a long substrate. 6. 6. The method according to claim 5, wherein the antibody is arranged on the inner surface of a capillary tube. Law. 7. The method according to claim 5, wherein the antibodies are arranged on a strip of paper. Law. 8. Claim 3, wherein the array consists of polymer beads having antibodies attached thereto. The method described in section. 9. the label is selected from the group of enzymes, radioisotopes or chromogenic substances , the method according to claim 1 or 2. 10. Claim 1 or 2, which qualitatively measures the unbound labeled binding substance. The method described in. 11. the label is an enzyme, and after contacting the test sample with said array of complementary binding substances. and adding to the collected test sample a substrate that changes color under the influence of said enzyme. According to claim 10, the unbound enzyme-labeled binding substance is measured by Method described. 12. an indicator that responds to the labeling of the unbound labeled binding compound; Measured by placing a substance adjacent to said array of crude complementary binding substances, whereupon contacting the test sample with the array of complementary binding substances and then contacting the indicator substance with the indicator substance; any unbound mark that comes into contact with the indicator substance in the test sample. Claim 10, wherein the presence of the indicator substance is indicated by the indicator substance. Method described. 13. Claim 1 or 2, wherein the unbound labeled binding substance is quantitatively measured. The method described in section. 14. the label is an enzyme, and after contacting the test sample with said array of complementary binding substances. the enzyme in the solution in addition to the collected test sample and a substrate for said enzyme. Claims that measure unbound enzyme-labeled binding substances by measuring activity. The method according to paragraph 13. 15. have mutually specific binding affinities and are composed of a binding substance and a complementary binding substance. to determine whether one of a pair of substances is present in a test sample in excess of a predetermined amount. A test kit comprising: a) A complementary binding substance that can bind to the binding substance and that is in contact with the test sample. are arranged on the surface of the touchable support and have a predetermined binding capacity for the binding substance. a complementary binding substance; and b) when added to said predetermined amount, substantially fills the binding capacity of said complementary binding substance; A test kit comprising a sufficient amount of a labeled binding substance. 16. Unbound labeled binding substance in the test sample after contact with said complementary binding substance The reagent according to claim 15, further comprising an indicator substance indicating the presence of the quality. test kit. 17. The complementary binding substance is an antibody, and the labeled binding substance is binding to the antibody. The test according to claim 15, which is a labeled antigen with specific binding affinity. kit. 18. The label is selected from the group of enzymes, radioisotopes or chromogenic substances. 18. The test kit according to claim 17. 19. The label is an enzyme and the indicator substance is a substrate that changes color under the influence of said enzyme. A test kit according to claim 16. 20. the support further comprises an indicator substance responsive to the label of the label-binding substance; wherein said indicator is arranged on said support adjacent to said complementary binding substance. The test kit according to item 15. 21. The label is an enzyme and the indicator substance is a substrate that changes color under the influence of the enzyme. , the test kit according to claim 20. 22. Substantially one-dimensional, underivatized antibodies with a predetermined number of antigen-binding sites a solid support on which an array of antibodies is immobilized, and a solid support adjacent to the antibody array that is specific for the antibody. It consists of a substrate for an enzyme that can bind to an antigen with a different binding affinity; An article whose color changes under the influence of a substance. 23. The solid support is longer and the antibody array and substrate are placed on the solid support longer. 23. The article of claim 22, wherein the article is arranged in the direction. 24. 23. The article of claim 22, wherein the support is a strip of paper. 25. The support is a capillary tube and the antibody and substrate are arranged on the inner surface of the capillary tube. 23. The article according to claim 22, wherein: