WO1995033205A1

WO1995033205A1 - Simple method and device for immunochemical assay

Info

Publication number: WO1995033205A1
Application number: PCT/JP1995/001006
Authority: WO
Inventors: Hideaki Manita; Kenichi Tajima
Original assignee: Teikoku Hormone Mfg. Co., Ltd.
Priority date: 1994-05-27
Filing date: 1995-05-25
Publication date: 1995-12-07
Also published as: AU2537895A

Abstract

The invention provides an assay method comprising an immunochemical qualitative or quantitative (semi-quantitative) analysis according to chromatography conducted by dissolving a marker serving as an index for judgement in a sample containing the object of analysis, allowing the sample and the marker to migrate to the site (detecting section) for reading the result of judgement on the chromatographic material, and visually judging the result of analysis, more specifically a simple immunochemical assay method (invention I) characterized by laminating a marker-containing member with a member (a marker elution accelerating member) made from a raw material that is equally coarse with or coarser than the marker-containing member. The invention also provides a simple semi-quantitative immunochemical assay method using a hapten as the object of analysis according to chromatography (invention II) including the indispensable constituent features of invention I and characterized by bringing (1) the object of analysis in a sample and (2) the marker-containing object of analysis or a chemical modification thereof into contact with (3) an antibody against the object of analysis, which antibody is immobilized on one and the same chromatographic material in various concentrations independently from the upstream side to the downstream side to thereby effect a competitive antigen-antibody reaction sequentially from the upstream side to the downstream side, and determining the concentration of the object of analysis in a sample by using the marker as an index. The invention further provides an assay device having such a construction as to accomplish the inventions I and II. According to the above method and device, it is possible in an immunochromatographic assay (qualitative or semi-quantitative analysis) to obtain clear and accurate assay results in a short time by allowing the sample and the marker to migrate on the chromatographic material synchronously. Further the invention provides an immunochemical method and a stick-shaped semi-quantitative device capable of conducting semi-quantitative analysis of a hapten-containing sample readily in a short time and clearly.

Description

Satsuki Itoda β Simple immunochemical assay method and device

The present invention relates to a simple immunochemical assay method and apparatus capable of easily and qualitatively or quantitatively (semi-quantitatively) clarifying and quantifying (semi-quantitatively) a sample containing an analyte in a short time. Background art

As a qualitative or quantitative method for trace substances contained in biological samples such as blood and urine, immunological measurement methods are widely used due to their high sensitivity. Of these methods, the so-called immunochromatography method using chromatography is easy to operate and requires only a short time for testing, so it is widely used in many situations today, such as clinical tests in hospitals and testing tests in laboratories. It is used.

The most common method of detecting a target substance in the immunochromatography method is to react a specific binding substance with various labels on a substance to be detected on a chromatographic material, and to detect a substance to be detected and a labeled specific binding substance. (Antigen-antibody complex) is formed and confirmed (detected) by various means. Labels include radioisotopes, chromophores, fluorophores, enzymes, colored particles, and the like. Examples of the detection means include a method using equipment such as a radiation detector and a spectrophotometer, and a method of visually confirming.

A qualitative analysis method by the immunochromatography method which can be visually judged and an apparatus therefor have been developed for various analytes, and many of them are commercially available and are generally used. One example is a pregnancy determination device.

The qualitative analyzer based on the immunochromatography method is generally based on the immunochemical San Germanti method and the competitive method. The configuration of the device is generally based on the site to which the sample containing the analyte is added (sample addition section) and the analyte. Sites containing labeling substances that serve as indices for detection (labeled sites), sites that capture and detect analytes (detection sites) And a part (absorber) that absorbs and removes excess sample.

Conventionally, to determine the amount of a substance to be detected in a sample, the sample containing the substance to be detected is appropriately diluted, a qualitative reaction is performed with a measurement reagent having a certain sensitivity, and the highest dilution ratio that indicates positive is determined. The semiquantitative value was calculated by multiplying the sensitivity, or a qualitative reaction was performed using reagents with different measurement sensitivities without diluting the sample, and the semiquantitative value was determined based on the sensitivity of the positive reagent. Methods for quantitative analysis include liquid-phase assays performed in containers such as test tubes and microtiter tubes, and solid-phase assays performed on chromatographic media.

Methods using reagents with different measurement sensitivities that do not require dilution of the sample have drawbacks such as difficulty in determination due to the difference in reaction strength at each measurement sensitivity, and are not widely used in practice.

The operation of diluting the sample increases the possibility that the tester will be infected with the pathogen from the test sample, such as blood or urine, in the field of medical treatment, especially in clinical testing. In addition, when semi-quantitative measurement is performed on a large number of samples, the operation efficiency can be significantly improved if no dilution operation is required.

Development of a quantitative (semi-quantitative) method and device using the immunochromatography method, which can be visually judged, is currently under development. For example, Japanese Patent Application Laid-Open No. Hei 4-351962 (corresponding European Patent Application Publication No. EP 516,095), Japanese Patent Application Laid-open No. Hei 5-57434, and international publication by the present applicant. There is a method or an apparatus described in W-94 / 28441. The type of the device is a so-called parallel arrangement of a plurality of samples with different concentrations (sensitivities) of analytes that can detect each unit consisting of the sample addition section, labeling substance existence section, one detection section and absorption section. A unit type (for example, Japanese Patent Application Laid-Open No. Hei 4-351926, International Publication No. WO94 / 28415 by the present applicant), and a sample addition part on one attrib A so-called stick type consisting of a part where a labeled substance is present, a plurality of detectors with different concentrations (sensitivities) of analytes (sensitivity) that can be detected, and an absorber (see, for example, JP-A-5-57) 4 No. 3).

Japanese Patent Application Laid-Open No. 4-351962 discloses a specific binding analysis method and apparatus capable of performing semi-quantitative analysis without diluting a sample. The method described in this publication is When qualitatively or quantitatively determining an analyte in a sample using the method of oral chromatography, a specific substance is allowed to exist in the measurement system, and the presence of the specific substance causes a labeled substance to be measured as an indicator of the analyte. The result is the same as diluting the sample containing the analyte (hereinafter referred to as dilution effect).

In the typical method described in this publication, the analyte added to the sample addition section is a specific substance which is not immobilized on the chromatographic material at a predetermined amount (concentration) and a specific substance present on the chromatographic material at a predetermined amount. Contact (in the specific binding substance existing area) with the labeled specific binding substance that is present without being immobilized. A certain amount of the analyte binds to the specific substance, but if the analyte is present in excess of the specific substance, the specific binding substance (specific substance or ^^ The part where the target substance can bind to the same binding site as the specific substance) is immobilized on the chromatographic material

(Detection unit). In the detection section, there is no site capable of binding to the specific substance, and at least the analyte bound to the specific substance passes through the detection section. At least only the analyte-labeled specific binding substance conjugate having a site capable of binding to the specific substance is immobilized in the detection section, and the immobilized conjugate is detected by various detection means. is there.

In the apparatus described in this publication, a plurality of assay strips having one detection unit on one chromatographic material were arranged in parallel by changing the setting of the concentration (sensitivity) of a detectable analyte in a stepwise manner. It is of the so-called unit type.

As a semi-quantitative method and apparatus in the unit format similar to the method described in Japanese Patent Application Laid-Open No. Hei 4-1351962, there is International Publication No. WO 94/28415 by the present applicant. . In this method, prior to the qualitative analysis of the analyte, each strip is treated with an antibody against the analyte that is immobilized in a stepwise different amount on each strip before the qualitative analysis of the analyte. Each time, a fixed amount of the analyte in the sample corresponding to the amount of the immobilized antibody is captured, and the concentration of the analyte in subsequent immunochemical qualitative analysis is reduced, and the analyte is detected A semi-quantitative value of the analyte corresponding to the amount of the capturing antibody in the strip at the limit or at the limit at which detection is not detected is determined.

Multiple detection sites on one assay strip instead of unit type The so-called stick type semi-quantitative method or device in which the analyte and the labeling substance in the sample react sequentially with these detectors by chromatographic transfer is a precedent example of clinical chemistry (CLIN. CHEM. 39/4). , 619-624 (1993)) and the method described in JP-A-5-5743.

The apparatus described in Clinical Chemistry uses lipoprotein (a), a complete antigen, as an analyte, and relates to a competition method using colloidal selenium-labeled lipoprotein (a). The device used has multiple measurement areas (detectors) on a single assay strip, and the most upstream detector has an anti-lipoprotein (a) that is immobilized on the downstream detector. The same amount of antibody is immobilized on all other downstream detection sections, with the same amount of antibody immobilized as the antibody.

JP-A-5-57443 discloses an immunoassay apparatus comprising a filter, a labeling reagent-containing member, and a porous carrier having one or a plurality of reaction regions, wherein the porous carrier is located upstream thereof. At the end, the filter is sandwiched between the filter and the labeling reagent-containing member, the reaction region is located downstream of the filter and the labeling reagent-containing member, and the labeling reagent-containing member contains the labeled reagent. There is disclosed a measurement device for measuring an analyte using a specific reaction, wherein a capture reagent is fixed in a reaction region. By immobilizing more capture reagents as the reaction area is located downstream, semi-quantitative analysis can be performed.

The sample addition section (sample application section) of the measurement device described in this publication has a labeling reagent-containing member arranged below the upstream end of the chromatographic material (porous carrier) as shown in c) of Fig. And a filter is arranged in the filter. By adopting such a structure of the sample addition section and the labeling substance existing section (labeling reagent-containing member), it is possible to quantify (semi-quantitatively) the analyte more sharply than the conventional method. The method described in this publication mainly uses a complete antigen as an analyte. In the conventional qualitative or quantitative (semi-quantitative) analysis method described above, the elution of the labeling substance from the site where the labeling substance exists may be slower than the flow rate of the added sample to the chromatographic material. Since the labeling substance does not move on the chromatographic material in synchronization, the pattern tends to be unclear when detecting the analyte. That is, the sample is analyzed Even after passing through the detection site of the target object, the labeling substance gradually elutes and moves to the detection site, so that the coloration at the detection unit becomes stronger over time, or the other than the detection unit on the chromatographic material Light color may be seen in some parts. These phenomena often make qualitative or quantitative (semi-quantitative) results uncertain.

In addition, by adopting a configuration such as the sample addition section of the apparatus described in Japanese Patent Application Laid-Open No. 5-57443, the results of the assay become clearer than in the case of the conventional apparatus, but the judgment pattern is still unclear. It leaves clarity.

Therefore, the present invention provides an assay by the immunochromatography method, in which a sample containing an analyte and a labeling substance for detecting the analyte are synchronously moved on a chromatographic material to obtain a clear and reliable assay result in a short time. The primary purpose is to provide a means for carrying out such tasks.

At medical sites where analysis by the immunochromatography method is always performed, it is desired to perform semi-quantitative analysis more easily, quickly, and with a smaller sample volume. Is preferred.

Japanese Patent Application Laid-Open No. 5-57434 discloses a semi-quantitative device in the form of a stick. However, this method mainly uses a complete antigen or an antibody against it as an analyte, and a hapten as an analyte. There is no specific description of the method. In addition, the method described in this publication has room for improvement in the clarity of analysis results (chromatographic patterns).

Therefore, the present invention provides a simpler, faster and more reliable (clear) determination result, and a semi-assay method using a stick-type immunochromatography method comprising a single assay strip capable of performing semi-quantitative determination of hapten with a small sample amount. A second object of the present invention is to provide a method for quantification, which can also achieve the first object of the present invention.

Further, a third and fourth object of the present invention is to provide an assay device having a configuration capable of achieving the first and second objects. Disclosure of the invention

The first object of the present invention is an immunochemical qualitative or quantitative (semi-quantitative) analysis by a chromatographic method, wherein a labeled substance serving as an index for determination is dissolved by a sample containing an analyte and the analyte is analyzed. Where the substance and the labeling substance read the judgment result on the chromatographic material In the Atsey method, including moving to the head (C)) and visually determining the analysis results;

A member containing a labeling substance (hereinafter, referred to as a labeling substance-containing member (b)) is a member made of a material having an eye roughness equal to or greater than that of the labeling substance-containing member (b) (hereinafter, a labeling substance elution accelerating member) (Referred to as "i")).

(Hereinafter, this method is sometimes referred to as "Invention I.")

The second object of the present invention is that, when a hapten is an analyte, the composition of the invention I further comprises (1) a hapten as an analyte in a sample, and (2) a labeled analyte. (3) Antibodies to the analyte immobilized on the same chromatographic material and the upstream of the chromatographic material at different concentrations at different concentrations from upstream to downstream. A simple immunochemical assay method characterized in that an antigen-antibody reaction is performed in a competitive manner sequentially from the downstream to the downstream, and the concentration of the analyte in the sample is determined using the label as an index.

(Hereinafter, this method is sometimes referred to as “invention II”.)

The third object of the present invention is to provide a sample addition section (A) for adding a sample containing an analyte, a specific binding substance to a labeled analyte, or a labeled analyte or a chemical thereof. (B) where the target denatured substance (labeled substance) is capable of chromatographic transfer, and one or more detectors (C) where the specific binding substance to the analyte is immobilized and present. ) And an immunochemical simplicity or quantitative (semi-quantitative) analyzer by a chromatographic method including an added sample and an absorbing section (D) for absorbing and removing a labeling substance not bound to the detecting section (C);

The labeling substance-containing member (b) constituting the labeling substance existing part (B) is laminated with the labeling substance elution accelerating member (i) made of a material having an eye roughness equal to or greater than that of the labeling substance-containing member (b). And a member (a) constituting the sample addition section (A) is brought into contact with the labeling substance elution accelerating member (i).

(Hereinafter, this device may be referred to as "Invention III.") The fourth object of the present invention is the above-described invention III, further comprising the step of: Device having a plurality of detectors (C on a single assay strip)

(Hereinafter, this method is sometimes referred to as "Invention IV.") BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a basic configuration of a semi-quantitative device (Invention III) having a configuration of a labeling substance existing portion (B) of the present invention.

FIG. 2 is a diagram showing the configuration of the device of the present invention (Invention IV) for the purpose of semi-quantitative determination of hapten.

FIG. 3 is an enlarged view of a labeled substance present portion (B) which is a characteristic portion of the present invention.

FIG. 4 is a diagram showing a configuration of a labeling substance existing portion (B) of a conventional semi-quantitative device. The reference numerals in the figure indicate the following.

A: Sample addition section

B: Labeled substance existing part

C: Detector

D: Absorber

a: Members constituting the sample addition section (A)

b: Label-containing material

d: Member of the absorption part (D)

e: Support

f: Double-sided adhesive tape

f and f 2: adhesive part

g: Chromatographic material

i: Labeling substance elution promoting member Best mode for carrying out the invention

Substances that can be analyzed in the qualitative or quantitative analysis by the method or apparatus of the present invention (Inventions I to IV) are roughly classified into complete antigens and haptens (incomplete antigens). Here, the complete antigen refers to an antigenic substance capable of inducing antibody production by itself (immunogenicity), and mainly includes peptide hormones having a large molecular weight. Haptens (incomplete antigens) are those that can bind to an antibody but do not have the ability to induce antibody production by themselves, such as peptides with relatively small molecular weight (molecular weight of about 1000 or less). included. The hapten obtains an antibody-producing ability when bound to a suitable carrier, for example, a protein such as serum albumin. Specific examples of these are shown below, but are not limited to those described here.

Examples of complete antigens:

(1) Examples of peptide hormones

1) Growth hormone (GH), adrenocorticotropic hormone (ACTH), melamine cell stimulating hormone (MSH), prolactin, thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), oxytocin Pituitary hormone

2) Calcium metabolism regulating hormones such as calcitonin and parathyroid hormone

3) Insulin, proinsulin, knee hormone

4) Gastrointestinal hormones such as gastrin and secretin

5) Angiotensin, bradykinin and other hormones that act on blood vessels

6) Placental hormones such as human placental gonadotropin (hCG) and human placental lactating hormone (hPL)

(2) Examples of other substances

1) Enzymes such as prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), alkaline phosphatase, transaminase, lactate dehydrogenase (LDH), transaminases, trypsin, and pebcinogen.

2) Cancer-specific substances such as human phytoprotein (AFP) and carcinoembryonic antigen (CEA)

3) Serum protein components such as immunoglobulin G (IgG), fibrin-fibrinogen degradation products (FDP, D-dimer), antithrombin II (ATIII), transferrin, etc. 4) Substances such as rheumatoid factor, serotonin, perokinase, ferritin, substance P, etc.

There are many other substances such as biological components and their metabolites.

Examples of haptens (incomplete antigens):

(1) Steroid type hub ten

1) Estrogen such as estrone, estradiol, estriol, estetrol, equilin, equilenin

2) Natural or synthetic progesterone such as progesterone, pregnanediol, pregnantriol, 19-nor-ethisterone and chlormadinone acetate

3) Male hormones such as testosterone, dehydroebiandrosterone, dihydrotestosterone androstene, etyocholanolone

4) Corticosteroids such as cortisol, cortisone, deoxycorticosterone, aldosterone, and tetrahydrocortisol

5) Bile acids such as vitamin D, cholesterol, cholic acid, deoxycholic acid and kenocholic acid, and other steroids such as cardiotonic steroids, saponins and sapogenins

(2) Bioactive amines

1) Potamines such as epinephrine, norepinephrine, dopamine, ephedrine and their metabolites

2) Morphine, codin, heroin, morphine hydrochloride, cocaine, mescaline, nopaverine, narcotine, yohimbine, reserpine, ergo-yumin, strikinine, etc.

3) Psychotropic drugs containing amino group such as LSD, amphetamine, methamphetamine, meprobamate

(3) Other examples

1) Non-antigenic low-molecular peptides such as TRH and LH-RH

2) Thyroid hormones such as jodothyronine, triothyronine, and thyroxine

3) Prostaglandin E2, Prostaglandin: E3, Prostaglandin F1 Prostaglandins such as

4) Vitamin A, B vitamins (vitamin B1, B2, B6, B12, etc.), vitamin E.

5) Antibiotics such as penicillin, actinomycin, chloromycetin, tetracycline, etc.

6) Other components existing in the living body, drugs administered to the living body, and metabolites thereof.

In the present invention, what can be a sample (specimen) is not limited as long as it contains the above-mentioned analyte, and mainly includes biological samples such as urine, serum, plasma, blood, saliva, amniotic fluid and the like.

The label that serves as an indicator of the qualitative or quantitative analysis of the analyte may be either a direct label or an indirect label. Direct labeling is preferred because it allows the assay results to be visually observed and does not require additional processing or steps. In the case of indirect signs, a process, process or device is required to visualize the sign after the completion of the atssay.

Labels that can be used for direct labeling include metal sols, colored latex particles, color indicators, coloring substances contained in ribosomes, dyes such as various dyes and pigments, and non-metallic sols such as carbon sol. , Luminol derivatives, chemiluminescent substances such as acridinium ester, and fluorescent substances such as fluorescein and rhodamine, but are not limited thereto.

Labeled substances that can be used for indirect labeling include, but are not limited to, various enzymes such as peroxidase, 3-galactosidase, alkaline phosphatase, perease, and glucose oxidase.

In the case of direct labeling, it can be obtained by visual observation of color tone, color density, luminescence intensity, fluorescence intensity, etc.In the case of indirect labeling, it can be obtained by changing the enzyme's substrate or chromogen depending on the enzyme used Detection is performed by measuring color density, luminescence intensity, etc.

In the present invention, the judgment of the analysis result by visual means that the direct and indirect targets are finally observed (visually) with the naked eye to determine the pattern of immunochromatography. Labeling substance in the present invention is a substance bound with the labeling, the type of analyte (or antigen or antibody, fully antigen or hapten or the like) different _c by, for example, the principle of the measurement method, the analyte is completely If it is an antigen and the measurement principle is the immunological sandwich method, the labeling substance is a substance that binds the above-mentioned labeling substance to a specific binding substance (for example, an antibody) for the analyte, and the analyte is a hapten (measurement The principle is a competitive method), for example, those in which the above-mentioned label is bound to the analyte hapten or a chemically modified product thereof.

Here, the chemically modified hapten is a chemical modification of the analyte hapten, which can competitively bind to an antibody against the analyte hubten in the presence of the analyte hapten. Hapten is a substance that is chemically modified. The chemically modified hapten and its modification method will be described later.

Further, the hapten which is a component of the labeling substance may be the hapten itself which is the analyte or the hapten which can bind to the detection antibody by a cross-reaction with the hapten which is the analyte. Good.

The labeling substance-containing member (b) of the present invention refers to a member in which the labeling substance is retained in a state where it can be chromatographed, and the retained labeling substance is dissolved by a sample solution containing the analyte and labeled. Move from the substance-containing member (b) to the chromatographic material, and move to the site (detection section (C)) where the qualitative or quantitative results are read together with the analyte. The labeling substance-containing member (b) is the same as the chromatographic material in which the detection section (C) is present, that is, the labeling substance-containing portion may be present on the chromatographic material, or may be a member different from the chromatographic material. However, it is preferable to use a member different from the chromatographic material. This is because the effect of the labeling substance elution accelerating member (i) described below can be more exerted.

In the present invention, the labeling substance elution accelerating member (i) refers to the labeling substance-containing member (b) that forms the labeling substance-presenting part by the capillary action of the sample solution containing the analyte added to the sample adding part. To increase the dissolution rate of the dried labeling substance impregnated in the labeling substance-containing member (b), speeding up the labeling substance to a state where it can be chromatographed, and synchronizing the analyte with the labeling substance. And has the function of assisting in chromatographic transfer, and is made of the same material as the labeling substance-containing member (b) or a coarser material. The labeling substance elution accelerating member (i) is a standard that indicates the coarseness of the filter media.

(mean flow pore size), a material of 10 / m or more is preferable, and a material of 30 to 5 is more preferable. As a material having an average flow pore diameter in the above range, glass fiber, woven fabric, non-woven fabric, or the like is preferable, and porosisilicate glass fiber is particularly preferable.

As the material of the labeling substance elution promoting member (i), it is necessary to use a material having an average flow hole diameter equal to or larger than that of the labeling substance-containing member (b) (coarse). The reason for this is that the sample penetrates uniformly and quickly into the laminated labeling substance elution promoting member (i), and the “labeling substance elution promoting member (i)” → “labeling substance containing member (b)” → It is considered that the flow of “chromatographic material” is promoted, and the labeling substance is redissolved from the labeling substance-containing member (b) —the chromatographic transfer is accelerated.

As a combination of the materials for the labeling substance elution promoting member (i) and the labeling substance containing member (b), the labeling substance containing member (b) is made of glass fiber, and the labeling substance elution promoting member (i) is made of cellulose filter paper or Glass fiber filter paper is preferred.

Further, the labeling substance-containing member (b) and the labeling substance elution accelerating member (i) may be made of the same material. In this case, both are preferably made of glass fiber, woven fabric or nonwoven fabric, and both are boroboro. It is particularly preferred that the silicate is glass fiber.

In this specification, “dissolution” in the case where “the labeling substance is dissolved” means that when the labeling substance is wetted by the sample, the labeling substance is in a state capable of chromatographic transfer. Next, in the method of the present invention, a semi-quantitative method (Invention II) when the analyte is a hapten will be described.

The haptens, samples, and the like to be analyzed in Invention II are as described in the above description.

The semi-quantitative method for hapten of Invention II is based on an immunochemical competitive reaction. That is, the analyte hapten in the sample and the labeling substance competitively bind to the specific binding substance (preferably an antibody) for the analyte immobilized in the detection section (C). Take advantage of that. Then, a plurality of detection units (〇! To から) composed of antibodies immobilized and present on the same chromatographic material are provided independently in order from upstream to downstream at a plurality of different concentrations, and each of these plurality of detection units (( ^ ~ ^) In the detection section (C), the hapten of the analyte in the sample and the labeled substance are subjected to antigen-antibody reaction in a competitive manner, and the coloring of the labeled substance appears according to the concentration of the analyte. Analyte concentration based on the number of

(Semi-quantitative value).

In the competitive reaction which is the principle of the present invention, the concentration of the analyte and the number of the detection units (C) detected (confirmed) by the label are inversely proportional. In other words, the higher the analyte concentration, the smaller the number of detectors (C) to be detected (confirmed).

For example, an antibody against the analyte hapten (hereinafter referred to as a hapten antibody) is immobilized as a detection substance on the detection section (C), and the hapten-carrier protein labeled as a labeling substance is bound on the labeling substance existing section (B). Or a carboxyl group-containing high molecular weight compound having a carboxyl group (hereinafter referred to as a labeled hapten-carrier conjugate) which is chemically bound to a product or a hapten or a chemically modified product thereof.

When a sample containing a certain concentration of the analyte is added to the sample addition section (A), the sample solution causes the label-hapten-carrier conjugate present in the labeling substance existing section (B) to elute and become ready for chromatographic transfer. . The label Haputen one carrier conjugate and analysis object, moves the detection unit by chromatography moved to (C), successively a plurality of detecting portions (C i~C _n) hapten antibodies present in different immobilized amount competing To combine.

In the competitive reaction method, the minimum concentration (detection sensitivity) of the analyte, which is capable of competitively inhibiting the binding reaction between the two, can be determined by the amount of the hapten antibody and the amount of the label-habten-carrier conjugate. Therefore, if different amounts of hapten antibodies are immobilized on multiple detectors (C to Cn) on the same chromatographic material, quantitative analysis with multiple levels of detection sensitivity can be performed on one chromatographic material. It can be done in one operation. How to set the detection sensitivity, that is, the range of the concentration (fixed amount) of the hapten antibody to be immobilized, should be appropriately selected depending on the analyte, the sample containing it, and the like. Further, in the plurality of detection units (C i C n), the concentration may be sequentially increased from upstream to downstream, or may be sequentially decreased, but the concentration may be sequentially increased from upstream to downstream. It is more preferable to comb. The reason for this is that the competition reaction occurs sequentially from the immobilized low-concentration hapten antibody upstream, so that the detection sensitivity can be increased and the detection concentration range can be narrowed.

Here, the hapten antibodies used in the present invention may be conventional antibodies (polyclonal antibodies) or monoclonal antibodies, and can be prepared by a known method. The hapten (analyte) or its chemically denatured product is bound to a substance having antigenicity such as serum serum albumin (BSA), and an antiserum is obtained by immunizing animals according to a conventional method using this as an antigen. Then, antibodies that are reactive only with the hapten (analyte) are separated from the obtained antiserum.

In the case of a monoclonal antibody, spleen cells of a mouse immunized with the above antigen and myeloma cells are fused, and a fused cell producing the desired antibody is selected to obtain a monoclonal antibody produced from the fused cell.

Further, hapten or a chemically modified product thereof, which is a component of a water-soluble monoolefin polymer containing a carboxyl group and a hapten-carrier protein-conjugated product or a hapten or a chemically modified product thereof chemically bonded thereto, are not competitive. Carrier protein or carboxyl group-containing water-soluble monoolefin polymer provides a binding site for a label or a chromatographic material to increase the reactivity with the corresponding hapten antibody. It fulfills.

Serum-derived proteins and ovalbumin (EA), such as rabbit serum albumin (BSA), rabbit serum albumin (RSA), goat serum albumin (GSA), and human serum albumin (HSA), are used as carrier proteins. be able to. When these are used, these proteins (for example, BSA) are used for the purpose of imparting antigenicity to haptens when producing hapten antibodies, and therefore, they have binding properties to these proteins. It is necessary to completely absorb and remove the antibody.

Chemical modification of the hapten is performed by chemically modifying the hapten so that it can chemically bond to the functional group of the water-soluble monoolefin polymer compound containing a carboxyl group (hereinafter referred to as spacer), for example, a carboxyl group or a hydroxyl group. Is added. Chemical modification can be performed by a known method according to the chemical structure of the hapten to be used. In particular, chemical modification to introduce carboxyl, amino and hydroxyl groups into hapten The method is preferred.

The spacer used in the present invention is a substance which is physiologically inert and generally does not have antigenicity. The spacer may have a hydroxyl group in addition to the carboxyl group, and these functional groups not only participate in the chemical bond with the hapten or its chemically modified product, but also in the polymer compound. It has the role of imparting water solubility to a certain spacer. Here, “water-soluble” of the spacer (water-soluble monoolefin polymer containing a carboxyl group) means that at least 1 part by weight of the spacer is completely dissolved in 1000 parts by weight of distilled water. To form a clear solution.

Spacer - average molecular weight of may be about 1 o ³ ~ 1 0 ⁷ or more, it is usually preferable of about several tens of thousand to several million.

Specific examples of the spacer include, for example, a home or copolymer of acrylic acid or methacrylic acid; a copolymer of maleic acid and vinyl acetate or a saponified product thereof; maleic acid and vinyl alcohol, for example, lower alkyl vinyl ether; And acrylic acid or a lower alkyl ester thereof, a copolymer with methacrylic acid or a lower alkyl ester thereof, or a hydrolyzate thereof. In addition

—A copolymer is, for example, a copolymer of acrylic acid or methacrylic acid and, for example, /?-Hydroxyethyl ester of acrylic acid or acrylamide, or a terpolymer having the above monomer as a structural unit. There may be.

The chemical bond between the hapten or its chemically modified product and the spacer is formed by an amide bond or an ester bond, and particularly preferably by an amide bond. When an amide bond is formed, for example, a known carpoimide method, a carbonyldiimidazole method, a mixed acid anhydride method, an active ester method, an azide method, an acid chloride method, and a diphenylphosphoryl azide (DPPA) method And carbodiimide method or DPPA method. Either of the above methods may be used, but some are unstable depending on the presence of a functional group that does not participate in the chemical bond of the hapten with the spacer. Should be avoided. The formation of the ester bond may be the case where the reactive hydroxyl group of the hapten or its chemically modified product is bonded to the carboxyl group of the spacer, and vice versa. In the former case, the carboxyl group of the spacer is converted into a reactive derivative, for example, an acid chloride. The reaction may be carried out with the hydroxyl group of the pentene, or with the hapten as it is if the spacer is a copolymer containing, for example, maleic anhydride. In the latter case, the principle of the method of forming an ester bond is the same as the former, but some haptens may not be stable enough to convert the carboxyl group into a reactive derivative, for example, an acid chloride. In this case, it is difficult to form an ester bond. As described above, in the semi-quantitative method for hapten (invention I), the use of the labeling substance elution accelerating member (i) enables quick and accurate (clear) semi-quantitative results to be obtained. Next, the device of the present invention (Inventions I and IV) will be described.

Next, the apparatus of the present invention will be described with reference to FIG. The device shown in a) of FIG. 1 is a basic example of the device of the present invention.

The apparatus of the present invention is a chromatographic type immunochemical qualitative or quantitative apparatus including a sample addition section (A;), a labeling substance existing section (B), a detection section (C), and an absorption section (D). The labeling substance elution promoting member (i) is laminated on the labeling substance-containing member (b) constituting the substance existing part (B), and the sample addition section (A) is further formed on the labeling substance elution promoting member (i). The members (a) to be formed are brought into contact with each other.

The device of the present invention (Invention I) is characterized by a specific configuration of the labeling substance existing portion (B), and the other portions and the portions added in addition to the above-described configuration of the present invention are particularly limited. There is no. The site other than the characteristic portion of the present invention or the site to be added differs depending on whether it is qualitative analysis or quantitative analysis, and the configuration corresponding to the analyte should be appropriately selected.

In addition, the device of the present invention can be applied to a so-called stick type or unit type as shown in FIG. 1 for quantitative (semi-quantitative) analysis, but the stick type device is more suitable for the present invention. The effect of is directly exhibited.

For the purpose of semi-quantitative determination of the hapten of Invention IV, as shown in FIG. 2, the apparatus of the present invention uses a sample addition section (A), a labeled substance presence section (B), a plurality of detection sections ((^ toス_ティ_ック_η ) and a stick type chromato-immunochemical analyzer sequentially having an absorption section (D). The device of the present invention shown in FIG. 2 is generally manufactured as follows. That is, attaching the double-sided adhesive tape (f) a support (e), to expose the adhesive portion and f _2. Place the upstream end of the chromatographic material (g) containing multiple detectors (C! To Cn) with an appropriate overlap on the adhesive, and fix it. A label-containing member with an appropriate overlap at its upstream end

(b) is fixed with an adhesive part. Further, the labeling substance elution promoting member (i) is placed on the upstream end side of the labeling substance-containing member (b) with an appropriate overlap, and the sample addition section is placed thereon.

Place the member (a) that constitutes (A), fix it with an adhesive part, and place the member (d) that constitutes the absorption part (D) with an appropriate overlap on the downstream end of the chromatographic material (g). fixed at f _2.

The components (A) to (D) constituting the device of the present invention shown in FIGS. 1 and 2 and the materials and the like of the components constituting the components will be sequentially described.

The material of the support (e) is plastic, glass, film, or the like, and should be appropriately selected depending on the type of the sample, the type of the analyte, and the like. The shape of the support (e) is preferably a rectangle with a length of 3 to 10 cm and a width of 0.5 to 2 cm. However, it depends on the type of the object to be analyzed, the number of detectors (C) set, the time of assembly, etc. It should be changed as appropriate.

The double-sided pressure-sensitive adhesive tape (f) is not particularly limited as long as it does not inhibit chromatosis, and commercially available tapes can be used.

The method of connecting the members constituting (A) to (D) is not particularly limited, and is not limited to an adhesive tape, and may be any method that can be connected.

In the apparatus of the present invention, the characteristic portion is the configuration of the labeling substance existing portion (B), and the other basic configuration is as described with reference to FIGS. 1 and 2 above. Fig. 3 shows an enlarged view of the labeled substance present area (B). Next, the configuration of each unit in the apparatus of the present invention (Invention III) for the purpose of semi-quantitative analysis will be described.

Sample addition section (A)

Examples of the material of the member (a) constituting the sample addition section include those having uniform characteristics such as cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth, but are not limited thereto. Not something. The sample addition section not only receives the sample containing the added analyte, but also has the function of filtering insoluble matter particles in the sample, etc., and therefore has a filtering function such as cellulose filter paper and glass fiber filter paper. Are preferred.

In order to prevent the analyte in the sample from non-specifically adsorbing to the material of the sample addition part during analysis, and to reduce the accuracy of analysis or semi-quantitative analysis, the material constituting the sample addition part must be prepared in advance. It is particularly preferable to use it after a non-specific adsorption prevention treatment. Examples of the non-specific adsorption prevention treatment include a treatment with an inactive protein, a treatment with a surfactant, and the like. The treatment with an inactive protein is performed, for example, using a material of 0.1 to 10% bovine serum albumin (BSA) 0.1M Tris buffer (pH 6 to 9) solution, 0.1 to 1 °% skim milk 0. Immerse in 1M Tris buffer solution (pH 6 ~ 9) solution and / or 0.1 ~ 10% zein solution, 37 ° C for 1 hour or 4 ° C-After leaving overnight, wash with Tris buffer and dry Become. The treatment with a surfactant comprises, for example, immersing the material in a 0.01% to 1% solution of Tween 20 or Triton X100, which is a nonionic surfactant, and drying it. Although it depends on the type of the analyte and the sample, it is preferable that the treatment with the inactive protein and the treatment with the surfactant are performed before use.

Labeling substance existing part (B)

The labeling substance existing part (B), which is a characteristic part of the present invention, comprises a labeling substance-containing member (b) and a labeling substance elution promoting member (i).

The material of the labeling substance elution promoting member (i) is as described above, and the treatment for preventing the analyte and the labeling substance from non-specifically adsorbing to the material constituting the labeling substance elution promoting member (i) is performed. After drying, make it.

The material constituting the labeling substance-containing member (b) used in the present invention can be the same as or different from the labeling substance elution promoting member (i) described above. Examples of the raw material include cellulose filter paper and Examples thereof include glass fiber filter paper, nonwoven fabric, and glass fiber. Preferred combinations of the components of the labeling substance-containing member (b) and the labeling substance elution promoting member (i) are as described above.

The labeling substance-containing member (b) is prepared by treating the analyte and the labeling substance to prevent non-specific adsorption, then impregnating a certain amount of the labeling substance and drying. For the purpose of semi-quantitative determination of hapten, the labeling substance-containing member (b) may be located on the same chromatographic material (g) as the detection unit (C), or it may be independent and different. It may be made of a material.

When the labeling substance-containing member (b) is located on the same chromatographic material (g) as the detection unit (C) described below, the detection substance is immobilized on the chromatographic material (g), and the analyte, the After the non-specific adsorption prevention treatment, apply the labeling substance to the upstream of the detection part (C) of the chromatographic material (g) and dry.

When the labeling substance-containing member (b) is made of a material different from the chromatographic material (g) in which the detection unit (C) described below is located, non-specific materials such as glass fiber, cellulose filter paper, glass fiber filter paper, and nonwoven fabric After the anti-adsorption treatment, a certain amount of labeling substance is impregnated and dried.

In the semi-quantitative method of the present invention, since the dissolution rate of the labeling substance and the uniformity of chromatographic transfer may affect the control of the measurement sensitivity, the labeling substance chromatographic material (g) Alternatively, the application or impregnation to the labeling substance-containing member (b) is preferably performed in the presence of a saccharide such as 0.1 to 10% mannitol or 0.1 to 30% saccharose.

The most important factor in accurately performing the semi-quantitative determination of hawthorn with the apparatus of the present invention (Invention IV) is that the analyte and the labeling substance in the sample are chromatographically moved in synchronization with each other, and both are detected by each detection unit ( (It is to arrive at ^ ~ (: ^) at the same time and perform a competitive reaction. That is, the elution of the labeling substance from the labeling substance existing part (B) is slow, and only the analyte is detected first. After reaching the part and reacting with the detection substance, the labeling substance is

When (Cl to Cn) is reached, the analyte hapten and the labeling substance, which are the measurement principles of the present invention, undergo incomplete competition reaction with the detection substance, and the result of the assay becomes uncertain.

In order to solve such a problem, as described above, the structure of the labeling substance existing portion (B) is configured such that the labeling substance elution promoting member (i) is laminated on the labeling substance-containing member (b). By promoting the elution of the substance and synchronizing and chromatographically moving the analyte in the sample and the labeled substance, the results of the assay can be made clear and reliable. Detector (c)

The detection section (C) is usually prepared by immobilizing a substance for detection on a part of the chromatographic material (g).

The method of immobilizing the substance for detection includes a method in which the substance for detection is directly immobilized on a part of the chromatographic material (g) by physical or chemical bonding, and a method in which the substance for detection is physically or There is an indirect immobilization method in which the fine particles are chemically bound and trapped in a part of the porous chromatographic material (g) to be immobilized. Either method can be used, but in the apparatus of the present invention, direct immobilization is preferred from the viewpoint of uniformity of insolubilization, ease of sensitivity adjustment, and the like.

The material (usually a chromatographic material (g)) constituting the detection section (C) is a porous nitrocellulose membrane, a porous cellulose membrane, a nylon membrane, a glass fiber, a nonwoven fabric, a cloth, etc. Those having an active group for binding a substance are preferable, and a porous nitrocellulose membrane and an activated nylon membrane are particularly preferable.

The shape of the substance to be detected immobilized on the chromatographic material (g) is not particularly limited, and may be any shape. However, the chromatographic material that enables uniform detection of the labeled substance with respect to the chromatographic tip. Particular preference is given to a line across (g).

The chromatographic material (g) is preferably used after immobilizing the substance for detection and then subjected to a non-specific adsorption preventing treatment such as a treatment with an inert protein.

In addition, when the semi-quantitative determination of the hapten of the invention IV is intended, a plurality of detection units (C i to

C n) is prepared by immobilizing the hapten antibody on the chromatographic material (g) at different concentrations sequentially from upstream to downstream.

Absorber (D)

The absorption part (D) is a part that absorbs and removes unreacted labeling substances that are not insolubilized in the detection part (C) while the added sample is physically absorbed by the chromatographic transfer. Cellulose filter paper, nonwoven fabric, cloth A water-absorbing material such as cellulose acetate is used. Since the chromatographic speed after the chromatographic tip of the added sample reaches the absorbing part (D) depends on the material and size of the absorbing material, the speed suitable for the measurement of the analyte can be determined by selecting it. Can be set.

The configuration of each part of the device of the present invention has been described above. The chromatographic speed (fluid flow speed) varies depending on factors such as the material, size, and thickness of the part. Therefore, one of these factors should be appropriately selected and set so that qualitative or quantitative analysis can be most suitably performed according to the type of the analyte and the measurement principle. The method and apparatus of the present invention can be applied to qualitative or quantitative (semi-quantitative) analysis by any immunochromatography method. By applying the present invention, clear and reliable analysis results can be obtained in a shorter time than before. Obtainable. Example

Hereinafter, the present invention will be described in more detail with reference to Examples. Example 1: Application of the present invention in semi-quantitative determination of urinary estrogen (hapten) by immunochromatography

1—a) Production of estriol—16—glucuronide BSA

Estriol one 16- Gurukuronai de was dissolved (hereinafter, E ₃ 16G hereinafter) (manufactured by Teikoku Seiyaku) 4 Omg dimethyl formamidine de 1. OML, this 4. After adding tri-n-butylamine 20. below C, isobutyl chlorocarbonate 11.21 was added and stirred for 30 minutes. Pre-added serum albumin

(Hereinafter referred to as BSA) (manufactured by Biocell) 117 mg dissolved in 2.8 ml of water, 1 NNaOH solution 15 was added, dimethylformamide 2. Oml was added, and the solution was kept at 8 ° C. Was mixed. This was then stirred at 8 ° C, added INNaOH solution 16. 6 l after one hour, further 3. After stirring for 5 hours, Sephadex G-25 in the unreacted E ₃ 16G and tri -n- Buchiruamin And other low molecular weight reagents were removed. After dialysis (against purified water) and lyophilization, Estrio 1-Ru 16-glucuronide BSA (E16 GBSA) was obtained. The co one bell reactions I a lyophilized powder of antigen binding of BSA1 mole per E ₃ 16G27~30 mol was confirmed.

1 production of single b) anti-E ₃ 16 G monoclonal antibody

The 1 was subcutaneously administered to an a) 3 week intervals BALB / C mice (6-8 weeks old) and E ₃ 16 GB S A25〃g produced with complete Freund indoor Juba cement, the outermost Later, 50 g was injected intravenously.

Three days after the final immunization, the spleen of the mouse was removed, spleen cells were collected, washed three times with Delpeco's modified minimal basic medium (hereinafter referred to as D'MEM), and the number of cells was counted. 2 × 10 ³ mouse myeloma cells were mixed with 1 × 10 ⁷ mouse myeloma cells P 3—NS—1 / 1-1 Ag4-1 (hereinafter referred to as NS-1) and centrifuged to collect the cells. A polyethylene glycol solution (PEG-1) warmed to 37 ° C was added to this pellet.

1% of 00: 4.25%, DMSO: 1.5% D'MEM) was added, and the centrifuge tube was slowly rotated for 1 minute to perform cell fusion. Add D'MEM at 37 ° C 10 times, 2 ml every 30 seconds, centrifuge, and pellet the pellet containing 20% fetal serum containing RPM.

1 -. 1640 as NS- 1 in medium 5 X 10 ⁴ cells / 0 were suspended so as to be 2 m 1, dispensed at 0. 2 ml in 96-well microphone port plate, and cultured in 5% C0 ₂ incubator Was. Twenty-four hours later, discard half of the supernatant from each well, add 0.1 ml of HAT medium (RPM-1640 medium containing hypoxanthine / aminopterin 'thymidine, 10% fetal calf serum), and then half-volume every 3 to 4 days Was cultured for 2 weeks while replacing the HAT medium, and the antibody activity of the culture supernatant in the grown well was measured.

The cells in which activity was observed were diluted in RPMI-1640 medium containing 10% fetal serum containing BALB / C mouse thymocytes and screened by limiting dilution to obtain 10 monoclonal antibody-producing fused cells. I got At least 2 × 10 ⁶ cells were intraperitoneally administered to BA LB / C mice to which 0.5 ml of pristane had been administered in advance, and ascites tumor was formed to obtain ascites. The ascites was purified by ammonium sulfate fractionation and Afigerupu Rotin A Mabusukitto to give the anti-E ₃ 16 G monoclonal antibody of white powder and freeze-dried.

1i c) Production of E 316 G—R S A

The 1-a) and E ₃ 16G and Usagi serum albumin (hereinafter in the same manner, using as RSA) (manufactured by Miles Inc.), was prepared E ₃ 16G- RSA.

1— d) Production of gold colloid-labeled E ₃ 16 G—RSA

E 316 G-RSA prepared in 1-c) was dissolved in purified water to prepare a 2 mg / ml solution. Colloids gold solution (gold colloids particle diameter 10 nm, manufactured by Amersham) after adjusted to pH 7. 6 8. to 0 ml by addition of _{_{0. 2MK 2 C0 3 30 ^ 1}} , E 3 16G — RSA solution 1001 was added, stirred at room temperature for 10 minutes, added with 40% 0.1% PEG600 solution, stirred for 10 minutes, and centrifuged at 15,000 rpm at 4 ° C for 60 minutes. After adding 4.0 ml of 0.1 M Tris buffer (pH 7.6) containing 0.3% BSA and 0.25% PEG 6000 to the obtained precipitate and suspending it uniformly, 1 After centrifuging at 5000 rpm for 60 minutes and repeating the same washing operation twice, 0.3% BSA, 0.25% PEG6000, 4% sucrose, and 0.2% l% NaN ₃ containing 0. 1M T ris buffer (pH 7. 6) was added to 0. 8 ml were uniformly suspended obtain gold colloids labeled E ₃ 1 60- 1¾3 eight solution, the labeled substance presence portion Stored at 4 ° C until preparation of (B).

1 -e) Production of sample addition section (A)

A filter paper for chromatography (No. 585, 0.85 mm thick, manufactured by Advantech Toyosha Co., Ltd.) was cut to make a 10 x 50 mm filter paper piece, which was then made into 5% skim milk powder (National Dairy Association). Contain 0.1 M Tris buffer (pH 8.2), immerse in a solution, incubate at 37 ° C for 1 hour, wash once with 0.1 M Tris buffer (pH 8.2), then 5% BSA (BioCell ) Contains 0.1M Tris buffer (pH 8.2). After incubating at 37 ° C for 1 hour, wash once with 0.1% BSA 0.1M Tris buffer (pH 8.2), drain, and then wash with 0.05% Tween 20, 0.1% BSA, 1% After immersion in a 0.1 M Tris buffer solution containing 0.1% sucrose, the solution was drained and freeze-dried, and used as a component (a) constituting the sample addition section (A).

1 -f) Manufacture of labeled substance present part (B)

(f-1) Preparation of labeling substance elution promoting member (i)

Borosilicate glass fiber (Borosilic at eGlass Fiber, average flow hole diameter: 39 j) (LYPORE, Grade 9254, manufactured by Lyda 11 companies) is cut into 10 x 50 mm strips. This is immersed in 0.1 M Tris buffer (pH 8.2) containing 10% nonfat dry milk37. C, after incubation for 90 minutes, washed twice with 0.1 M Tris buffer (pH 8.2), and immersed in 1 M Tris buffer (pH 8.2) containing 5% BSA. After incubation at 37 ° C for 1 hour, put in 0.1 M Tris buffer (pH 8.2) containing 0.1% 83. After soaking, the solution was drained and dried at room temperature, and used as a member for preparing a labeling substance elution promoting member (i) and a labeling substance-containing member (b).

(f-2) Preparation of labeling substance-containing member (b)

The gold colloid-labeled Eal 6G-RSA solution prepared in 1-d) was diluted with the same amount of a 0.1% NaN ₃ solution containing 30% sucrose, and the 10 x 20 mm solution obtained in (f-1) above was obtained. The material was impregnated with 1201 and freeze-dried to prepare a labeling substance-containing component (b). This was cut into 5 x 2 mm when the semi-quantitative device was prepared, and used for a 5 x 5 mm labeling substance elution accelerating member (i) sandwiched from above and below by two (11) h) described later.

1— g) Manufacture of detector (C)

Nitrocellulose membrane (chromate material (g)) (Sartorius, pore size 8 zm) is cut into a size of 30 x 100 mm, and 1-b) using Cammag Linomat IV 0.15, 0.2, 0.3, and 0.4111 / 1111 solutions (50 // 111183 containing 5 OmM Tris buffer, pH 8.2) of the anti-E16G monoclonal antibody prepared in 10 // 1 was sprayed linearly in the order of 12, 14, 16 and 18 mm from one end, left in a thermo-hygrostat at 25 ° C and 80% humidity for 25 minutes, and then 0.1M Tris buffer (pH8) 2) In 5% skim milk powder (National Federation of Dairy Federations) dissolved, then dipped in 5% BSA solution and blocked, then 0.1% Tris buffer containing 1% saccharose and 1% mannitol The cells were washed, allowed to stand at room temperature, and dried to form a detection section (C) containing four detection sites.

1 -h) Production of semi-quantitative equipment

1-g) The nitrocellulose membrane containing the detection section (C) manufactured in 1-g) was cut into 100 mm wide strips of 5 mm width to form a 5 x 3 Omm strip (hereinafter the detection section (C) fixed side with low antibody concentration) Is called upstream). The piece of filter paper for the sample addition section (A) manufactured in 11e) was also cut to make a strip of 8 x 1 Omm.

As it is shown in a) of FIG. 1, attaching the double-sided adhesive tape one 7 ° (f) brass tic plate as a support (e), in the figure: exposing the adhesive surface of the f ² parts. Part of chromatographic material (g) Labeling substance elution promoting member (i) (5x5mm) manufactured in 1-f) on contact side Then, the upstream side of the strip (chromatographic material (g)) (5x10 mm) of the nitrocellulose membrane containing the detection unit (C) manufactured in 1-g) above is labeled

It was placed on the plastic plate (E) so that it overlapped (i) by 2 mm. Further, the labeling substance-containing member (b) is overlapped with the chromatographic material (g) so that the upstream end of the chromatographic material (g) overlaps the labeling substance-containing member (b) (5x2 mm) manufactured in 1-f) by lmm. ) And another labeling substance elution accelerating member (i) so as to sandwich the labeling substance-containing member (b). On top of that, a strip of filter paper (8x10m m) was placed thereon and fixed with the adhesive portion of the double-sided adhesive tape (f) (FIG. 1, d). Finally, a piece of 8x15 mm filter paper of chromatography filter paper (Advantech Toyo, No. 585) is used as the absorption part (D), and the downstream part of the strip of the nitrocellulose membrane (chromatographic material (g)) in the detection part (C). The double-sided adhesive tape (f) was fixed to the adhesive surface of the adhesive portion f2 with an overlap of 3 mm from the end.

Measurement

(i-i 1) Measurement by male urine

0 E ₃ 16 G in Esutorio Ichiru terms, 12.5, 25, so that the respective concentrations of 50 and 10011 / ml dissolved in male urine samples were prepared. This sample was tested by adding 100 1 of each concentration of feed to the sample addition section (A) of the semi-quantitative device prepared in 11 h).

Esutorio Ichiru in the sample compete sequentially from upstream in the movement detection unit to (C) by chromatography move with dissolved gold colloids labeled E ₃ 16 GR SA by the sample and the downstream fixed anti E ₃ 16 G monoclonal antibody After the unreacted label moved to the absorption part (D), a colored band was observed at the detection part (C).

Detection sensitivity of the apparatus, by a labeling substance present section (B) in the gold colloids labeled E 316G- each anti E ₃ 16 G monoclonal antibody immobilized to the holding amount and a detection portion of the RSA (C), both The reaction was set so that it could be blocked by 12.5 ng / ml in the uppermost detection unit and 25, 50, and 100 ng / ml in the downstream detection unit.

Table 1 shows the results of this quantitative measurement. In Table 1, column (a) shows the number of colored bands in the detection part (C), and column (b) shows the case where band formation was observed (+). The judgment pattern in which the case was not shown is indicated by (1). Table 1 S (a) (b)

ng / m color band judgment pattern

0

12.5 +

25 ++

50 one + + +

100 + + + +

The results in Table 1 show that semiquantitative results (patterns) were obtained according to the detection sensitivity set for each sample.

The time from sample addition to completion of Atsushi was about 3 minutes. After that, no change was observed in the judgment pattern even when left unattended.

(i-i 2) Measurement by women's urine

Next, the number of color bands observed in column 2 (a) and the color bands formed in column (b) are shown in Table 2 (a) for three cases of normal female urine collected in each period of the menstrual cycle (+ ), The determination pattern is shown as (1) if not formed, (c) the semi-quantitative value by this method, and (d) the quantitative value by Radioimnoassay (RIA). Was. Table 2

(a) (b) (c) (d)

Female urine coloration; point "Judgment 8 ° turn Semi-quantitative value (ng / ml) RIA value (ng / ml) Follicular phase (A) + 12.5 13 8

(B) 12.5 or less 7 2 (C) + 12.5 15 1 Ovulation period (A) 50 65 0

(B) + + + + 100 94 7

(C) + + + + 100 105

Luteal phase (A) —— + + 25 23.3

(B)-+ + 25 20.2

(C) + 12.5 17.4

From the results in Table 2, it can be seen that the semi-quantitative value by this method is in good agreement with the quantitative value by RIA. The measurement time for the above semiquantitative analysis using feminine urine as in (i-1) was 3 minutes, as in the case of male urine (i-1), and no change was observed in the judgment pattern even after standing. Example 2: The semi-quantitative determination of pregnanediol (Habten) in urine

Application examples

2 -a) Production of Pregnandio One-Ru 3-Glucuronide BSA

P-dio13G-BSA was produced in the same manner as in 11a) of Example 1 using pregnanediol-13-glucuronide (hereinafter referred to as P-diol3G) and BSA.

2—b) Production of anti-P—dio 3G monoclonal antibody

Using P-dio 13G-BSA produced in 2-a) above, 11-b) of Example 1 In the same manner as described above, four anti-P-dio13G antibody-producing fusion cell lines were obtained. For the monoclonal antibody, 2 × 10 ⁶ cells were transplanted into the abdominal cavity of a BALB / C mouse to which 0.5 ml of pristane had been intraperitoneally administered in advance, and ascites was collected, and the ascites was collected using P-diol 3G-BSA. Purification was performed by affinity chromatography using the bound Sepharose 4B (Pharmacia) to obtain an anti-P-diol 3G monoclonal antibody.

2-c) Preparation of pregnanediol-l-glucuronide-bonded vinyl methyl ether anhydrous maleic acid copolymer

(c-1) Production of P-diol 3G-lysine derivative

P-diol 3G99mg, p-benzyloxycarpoyllysine methyl ester. Toluenesulfonate 14 Omg was dissolved in DMF 12ml, and the mixture was stirred under ice-cooling while diphenylphosphoryl azide 65mg, then triethylamine 0.05. After adding 6 ml, the following structural formula was obtained in the same manner as in 1-b) of Example 1.

The desired P-dio 13 G-lysine derivative represented by the following formula (10 Omg, relative to theoretical yield of 58%) was obtained.

(c-2) Production of P-dio 3 G-linked vinyl methyl ether maleic anhydride copolymer

Dissolve 38 mg of P-di013G-lysine derivative obtained in (c-1) above in 3 ml of methanol, add 1 Omg of palladium black, and stir in a steam stream at normal temperature and normal pressure. You. The reaction was completed in 2 hours, the catalyst was removed by filtration, the filtrate was concentrated under reduced pressure, and dimethyl ether was added to the residue to obtain 26.6 mg of the desired product as a powder. This product was dissolved in 6 ml of DMF (dimethylformamide) with 100 mg of vinyl methyl ether maleic anhydride copolymer (hereinafter referred to as PVMMA) under heating, and then DCC (dicyclohexyl carpoimide) was added. 4 mg was added and left at room temperature for 4 days. The reaction solution was dialyzed, and a 30 ml solution of Pdi013G-bonded vinyl methyl ether maleic anhydride copolymer (hereinafter referred to as P-diol 3G PVMMA) with a pregnanediol content of 0.46 mg / ml (determined by sulfuric acid coloring) I got

2— d) Production of colored latex label P—d i 01 3 G PVMMA

Red aminated polystyrene latex (solid content: 10%, manufactured by Nippon Synthetic Rubber Co., Ltd., particle size: 0.37 // m) 0.5 ml of N, N-dimethylformamide (DMF) 'purified water (1: 1) 12 ml of the prepared 10% triethylamine solution was added and suspended. After stirring for 15 minutes, the mixture was centrifuged. The precipitate was centrifugally washed twice with 10 ml of DMF / water (1: 1) and once with 10 ml of purified water, then suspended in 0.5 ml of purified water, and P-diol prepared in 2-c) To 0.63 ml of 3G PVMMA solution (equivalent to 0.29 mg of pregnanediol), add and mix 1 ml of purified water, and then add 7.5 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. The reaction was carried out overnight with stirring. After the completion of the reaction, the precipitate obtained by centrifugation was repeatedly centrifuged and washed three times with 10 ml of glycine buffer, and then 30% saccharose and 0.1% goat serum albumin (hereinafter referred to as GSA) (Miles Co., Ltd.). The suspension was suspended in 10 ml of a glycine buffer solution to produce P-diol 3 G PVMMA-bound colored latex.

2-e) Production of sample addition section (A)

A filter paper for chromatography (No. 526, manufactured by Advantech Toyo Co., Ltd.) was cut into a piece of filter paper of 10 × 50 mm, and the sample-added part (A) was manufactured in the same manner as in Example 11 e). .

2—f) Manufacture of labeled substance present part (B)

(f-1) Manufacture of a member for producing the labeled substance-containing member (b) and a member for promoting the elution of the labeled material (i)

Borosilicate glass fiber filter paper (Boros ili cat e G 1 a ss Fiber (average pore diameter: 35 m) (L YPORE, GRADE 9818, manufactured by Lydal 1) cut into 10 x 50 mm strips and treated in the same manner as in Example 1-f) Thus, a member for producing the labeling substance-containing member (b) and a labeling substance elution promoting member (i) were obtained.

(f-2) Manufacture of labeling substance-containing members (b)

The labeled latex-labeled P-dio 13 G PVMMA suspension prepared in 2–d) was cut into a 10 x 20 mm suspension to produce the labeled substance-containing member (b) obtained in (f-11) above. It was impregnated with 120〃1 and dried at room temperature. This was cut into 5 x 2 mm when the semi-quantitative device was manufactured to obtain a labeling substance-containing member (b), and a 5 x 5 mm labeling substance elution promoting member (i) was laminated and used in 2-h) described later.

2-g) Production of detector (C)

Nitrocellulose S-mo (ce 11 u10 set rate on My1ar, pore size 8 / m, manufactured by Zaritrius) is cut into a size of 30 × 100 mm, and cut with a force mag. 0.5, 1.0, 1.5, and 3 mg / ml solutions (5 OmM Tris buffer containing 50 µg / ml BSA, pH 8.2) of the anti-P-dial 3G monoclonal antibody prepared in b) 1 was linearly sprayed sequentially at positions 12, 14, 16 and 18 mm from one end, and processed in the same manner as in Example 11-g) to form a detection section (C) having four-stage detection sensitivity. Produced. The nitrocellulose membrane (cellulose nitrate on Mylar) used here is a nitrocellulose fiber directly molded on a plastic plate. By using this, distortion due to expansion of the chromatographic material due to wetting by the sample solution can be prevented.

2—h) Production of semi-quantitative equipment

Cut the 100mm-wide Nitrocell orifice (chromatographic material (g)) including the detector (C) manufactured in 2—g) into 5 mm width and cut it into a 5 X 30 mm strip. The prepared filter paper piece for the sample addition section (A) was also cut into a strip of 8 × 10 mm. As shown in Fig. 1 b), a double-sided adhesive tape (f) is applied to the entire surface of one side of the plastic plate as the support (e), and the downstream end 12 mm of the double-sided adhesive tape (f) is left thereon. A strip of nitrocellulose membrane (chromatographic material (g)) containing the detection section (C) (5 x 30mm) (the right end is the upstream end in b) in Fig. 1). Next, contacting the upstream end of the chromatographic material (g), the labeling substance elution accelerating member manufactured in 2-f)

(i) Paste (5x5mm), and the downstream end of the labeling substance-containing member (b) (5 x 2mm) manufactured in 2—: f) is the upstream end of the nitrocellulose membrane (chromat (g)) and the labeling substance. Place the elution-promoting member (i) so that it overlaps with the downstream end by 1 mm, and place another labeling-elution-promoting member (i) (5 x 5 mm) on top of it. A strip of filter paper (8x10mm) was placed on the tape and fixed with the adhesive at the upstream end of the double-sided adhesive tape (f).

Finally, a nitrocellulose membrane (chromatographic material (g)) containing an 8 x 15 mm filter paper piece from a filter paper for chromatography (Advantic Toyo, No. 585) as the absorbing part (D) and containing the detecting part (C) Double-sided adhesive tape with 3 mm overlap at the downstream end of

It was fixed with the adhesive part on the downstream end side of (f) (d in Fig. 1).

2— i) Measurement

Pregnanediol in urine was measured in 7 cases of urine from pregnant women. Pregnant women's urine was added at 150/1 to each sample addition section (A) of the semi-quantitative device prepared in 2-h), and the measurement was performed. In this semi-quantitative measurement, the detection sensitivity was 2.5 g / ml or less for four color bands, 5 / g / ml for three bands, 10 /// !! 11 for two bands, and 20 // for one band. It was set to be 40 g / ml or more for 1111 and 0 tubes.

Table 3 shows the results. In Table 3, (a) the number of color bands observed, (b) the judgment pattern in which a color band was formed (+) in column (b) and (-) if no color band was formed in column (b). Column (c) shows the semi-quantitative value, and column (d) shows the quantitative value of pregnanediol in the sample by Radioimnoassay (RIA).

Table 3

(a) (b) (C) (d) Pregnant woman urine colorant ', Judgment c. Turn semi-quantitative value (ng / ml) RI weave (ng / ml)

1 1 1 1 + 5 6.3

2 1 1 —— + + 10 11.0

3 20 20.5

4 1 1 1 1 2.5 or less 1.3

5 + 5 5.5

6 1 1-+ + 10 12.0

7 40 or more 60.5

The results in Table 3 show that the semiquantitative value of pregnanediol in each pregnant woman's urine is in good agreement with the quantitative value by RIA. The semi-quantitative measurement time of the present example was approximately 3 minutes, and there was no change in the judgment pattern even after being left as it was. Example 3: Application of the present invention in qualitative analysis of urinary hCG (human placental gonadotropin)

3— a) Production of anti-hCG monoclonal antibody

HCG (10000 iu / mg) was administered subcutaneously to the B a1 b / c mouse three times at three-week intervals together with complete Freund's adjuvant, and three weeks later, hCG was intraperitoneally administered. Cell fusion between spleen cells and myeloma cells (NS-1) 3 days after the final immunization is performed by a conventional method. After HAT selection, cloning is repeated, and a fusion cell line secreting hCG-specific antibodies, hCG, hLH, hF A fusion cell line secreting a monoclonal antibody that recognizes the Hi subunit that cross-reacts with SH was obtained.

Each cell line was administered intraperitoneally to B a1 b / c mice to which pristane had been administered in advance, and ascites tumor was formed to obtain ascites. The ascites obtained was subjected to ammonium sulfate fractionation and Affigel-Pro Purification was performed using a Tin A Mabs Kit, followed by lyophilization to obtain a white powdered monoclonal antibody.

The obtained anti-hCG-specific monoclonal antibody was used for the action of the detection part (C), and the monoclonal antibody recognizing the single subunit was used for producing a labeled antibody.

3-b) Preparation of anti-mouse gamma globulin (yG) antibody

1 mg of Mouser G (manufactured by Miles) was dissolved in 1 ml of physiological saline, emulsified with the same amount of Complete Freund's adjuvant, and injected subcutaneously into the feet of adult rabbits. This injection was performed three times at one-month intervals. After confirming an increase in antibody titer, whole blood was collected to obtain antiserum. This antiserum was inactivated at 56 ° (30 minutes, then salted with ammonium sulfate, purified by DEAE-cellulose chromatography, and gel-filtered with Sephadex G200 (Pharmacia), and then lyophilized to a white powder. Anti-mouse G antibody was obtained.

3—c) Production of anti-hCG monoclonal antibody labeled with gold colloid

The anti-hCG monoclonal antibody (antibody recognizing hydsubunit) prepared in 3-a) above was dissolved in Tris buffer (pH 8.2) containing BSA55 / zg / ml, and dissolved in 20 O ^ g / A ml solution was prepared. Colloids gold solution (gold colloids particle size 10 nm, Amashiamu Co.) After adjusting the p H 7. 6 by addition of 0. 2mK ₂ C_〇 ₃ 15 1 to 4. OML, the antibody solution 0. 5 ml After adding and stirring at room temperature for 10 minutes, 2% 0.1% PEG 6000 solution was added, and after stirring for 10 minutes, 15000 rpm, 4. C, centrifuged for 60 minutes. To the resulting precipitate, add 0.1 M Tris buffer (pH 7.6) containing 0.3% BSA and 0.25% PEG6000, add 2. Oml, uniformly suspend, and centrifuge at 15000 rpm for 60 minutes. After repeating the same washing operation twice, 0.1 M Tris buffer (pH 7.0) containing 0.3% BSA, 0.25% PEG6 000, 4% sucrose, and 0.05% thimerosal was added to the obtained precipitate. .6) 0.4 ml was added, and the mixture was uniformly suspended to obtain a gold colloid-labeled anti-hCG monoclonal antibody solution, which was stored at 4 ° C until preparation of the labeled substance-presenting portion (B). 3-d) Production of sample addition section (A)

The member (a) constituting the sample addition section (A) prepared in Example 1-e) was used. 3-e) Production of (B) in the part where the labeling substance is present (e-1) Preparation of labeling substance elution promoting member (i)

Example 1 A labeling substance prepared by treating a porous silica glass fiber (average pore diameter: 39 jm) (LYPOREs Grid 9254, manufactured by Lydal 1) with f) and (f-1) Elution promoting member (i) was used.

(e-2) Preparation of the labeling substance-containing member (b)

Example 2—Porosilicate glass fiber (average pore diameter: 35 im) (LYPORE, Grade 9818, manufactured by Lydal 1) was manufactured in f) and (f-1). The member was used for producing a marker-containing member (b).

Gold colloids labeled anti-hCG monoclonal antibody solution prepared in 3- c) was diluted with 30% Shiyukurosu containing 0. 1% NaN ₃ solution the same amount, frozen by 1 20/1 impregnated into the member of 10 x 20 mm After drying, a labeling substance-containing member (b) was prepared. This was cut into 5 x 2 mm when the qualitative apparatus was manufactured, and a 5 x 5 mm labeling substance dissolution promoting member (i) was laminated and used in 3-g) described later.

3-f) Manufacture of detector (C)

Nitrocellulose membrane (chromate material (g)) (Sartorius, pore size 8〃m) is cut into a size of 30 x 100 mm, and is then cut using Cammag Linomat IV 3-a) Of a 0.5 mg / m 1 solution (50 mM / g / m IBSA-containing 50 mM Tris buffer, pH 8.2) of the anti-hCG specific monoclonal antibody prepared in Step 1 from one end of the chromatographic material (g) At the position of 10 mm, then use 10% of the 2 mg / ml solution of the anti-mouse G ゥ sagi antibody prepared in 3-b) (5 OmM Tris buffer containing 50 zg / ml BSA, pH 8.2) as a control. After linearly spraying 17 mm from one end of the chromatographic material (g), leave it in a thermo-hygrostat at 25 ° C and 80% humidity for 25 minutes, and then proceed as in Example 11g). After performing blocking treatment and drying, a detection part (C) was prepared.

3 -g) Manufacture of equipment for qualitative analysis

The width of 100 mm of the nitrocellulose membrane including the detection unit (C) manufactured in 3-f) was cut into 5 mm width to form a 5 x 30 mm strip (hereinafter, the anti-hCG antibody fixed side is referred to as upstream). In addition, a piece of filter paper for the sample addition section (A) manufactured in 3–d) was also cut to produce an 8 × 10 mm strip. As shown in Fig. 1 c), a double-sided adhesive tape (f) is applied to the entire surface of one side of the plastic plate, which is the support (e). A strip (5 x 30 mm) of a nitrocellulose membrane (chromat (g)) containing (C) was applied (the right end is the upstream end in c) in Fig. 1). Next, on the upstream end of the chromatographic material (g), the labeling substance-containing member (b) (52 mm) manufactured in 3-e) is placed, and on top of this, the labeling substance elution promoting member manufactured in 3-e) (I) (5x5 mm) was laminated along with the left end of the labeling substance-containing member (b), and the right side was fixed with the adhesive surface of double-sided adhesive tape (:). Further, a strip of filter paper (8 × 10 mm) for the sample addition section (A) was placed thereon, and fixed with the adhesive section at the upstream end of the double-sided adhesive tape (f). Finally, an 8 × 15 mm piece of filter paper for filter paper for chromatography (Advantech Toyo, No. 585) is used as the absorbing part (D) as a bitrocellulose membrane (chromatographic material (g )) Was fixed to the downstream end of the double-sided tape (f) with a 3 mm overlap on the downstream end of the double-sided tape (f) (Fig. 1d).

3— h) hCG measurement

In order to compare the qualitative analysis performances due to the difference in the configuration of the labeling substance existing part (B), 3-g) was prepared with 3-g) except that the labeling substance elution promoting member (i) in the apparatus of the present invention was not used. A device for qualitative analysis similar to that described above was prepared and used as a control.

Samples containing 0, 10, 25, and 5 Omiu / ml of hCG were prepared in male urine, and each sample 1501 was added to the sample-added part (A), and the formation of a colored band was observed over time. . Table 4 shows the results.

Table 4 Judgment time hCG (miu / ml)

(Min) 0 10 25 50

1 ± + +

Book 2 + + +

Departure 3 + + +

Akira 5 + + +

10 + + +

1

Vs. 2

3 Satoshi 5 + Sat +

10 ++

I: No colored band

Soil: slightly colored band formation

+: Clear color band formation From the results in Table 4, with the configuration of the labeled substance present portion (B) of the present invention, formation of a color band was observed even at 10 miu / ml 1 minute after the addition of the sample. A clear color band was formed in 2 minutes. In Omiu / ml, in contrast while color band is not formed in a _c-after 10 minutes, slow the appearance of color bands, the 10 mIU / ml, exhibit only slight coloration even 5-10 minutes, 25Mi u A clear color band was observed at 10 minutes at 50 ml / ml and at 5 minutes at 50 miu / ml.

As described above, even in the qualitative analysis test, a highly sensitive and highly accurate result was obtained in a short time, and the effect of the present invention was remarkably recognized. Example 4: Comparison of semi-quantitative performance (clarity and accuracy) depending on the difference in the composition of the labeled substance presence part (B)

Comparison of semi-quantitative performance (clarity and accuracy of the judgment result) due to the difference in the configuration of the labeling substance existing part (B) was performed using the estrogen measurement of Example 1 as a model. The configuration of the labeled substance present part (B) of the present invention is the same as that of the _Q comparative example (B) shown in a) of FIG. 1 and a) of FIG. (A), b), and c) in Fig. 4 (The configuration in Fig. 4 is one generally used for qualitative immunochromatography such as urinary human placental gonadotropin (hCG). ). The semi-quantitative apparatus of the comparative example uses the same raw materials (materials) as in Example 1 in order to prevent the difference between the raw materials (materials) of the sample addition section (A) and the labeled substance presence section (B). Figure 4 shows a) b) and c).

Table 5 shows the time course of the reaction pattern of the samples with estrogen concentrations of 0, 12.5, 25, 50 and 100 ng / ml and the observation results.

Table 5: Jest D ', Judgment Sample addition part (A)-Constitution of labeling substance existing part (B) Concentration Time Comparative example of the present invention

(ng / ml) (min) Figure a) Figure 3-a) Figure 3-b) Figure 3-c)

2 ++++ ++++ + + + +

0 3

Five

2 —— f- ±±±± Sat ±±± ±±±

2. 5 3-+ 11+ + 1+ + 1+

5-+ Sat + +

2 ++ soil ++ ±± ++ ±± ++

25 3 ++ ― ±± + one ±± + — ++

5 ++ —one ±± one ±± —one ± +

2 ±± ++ Sat +++ +++

50 3

5 Elder + Elder + h +

2 + + + + + + + + + + + + + + + +

00 3 + + + + ±± + + ±± + + + + + +

5 + + + + ± ± ± + ± ± ± + ± ± + + 1: clear color band formation

Shi: Slightly colored band formation

+: No color band formed (blocked) In the configuration of the labeled substance present portion (B) of the present invention, the presence or absence of the formation of a color band became clear 2 minutes after the addition of the sample, and no change was observed in the determination pattern even after 5 minutes. In each of the configurations (a, b, and c in Fig. 4), the presence or absence of a colored band is unclear at 2 minutes after sample addition, and the colored band becomes clear over time. In the detection part (C) where no band should be formed, a slight color band or a clear color band was observed.

As described above, the judgment pattern of this method was extremely clear and highly accurate. The invention's effect

According to the method or apparatus of the present invention, in a qualitative or quantitative (semi-quantitative) analysis by the immunochromatography method, a sample containing an analyte and a labeling substance for detecting the analyte are synchronized and the chromatographic material is placed on the chromatographic material. It can be moved to get clear and reliable results in a short time.

The method or apparatus of the present invention provides a so-called stick-type immunochemical method and apparatus capable of easily, clearly, and semi-quantitatively determining a sample containing the hapten to be analyzed in a short time, in a short time.

Claims

Scope of demand

1. In immunochemical qualitative or quantitative (semi-quantitative) analysis by the chromatographic method, the labeling substance used as a judgment index is dissolved by the sample containing the analyte, and the analyte and the labeling substance are placed on the chromatographic material. In an Atsey method including moving to a site (detection unit (C)) for reading the judgment result of (1) and visually judging the analysis result;

A member containing a labeling substance (hereinafter referred to as a labeling substance-containing member (b)) is a member made of a material having an eye roughness equal to or greater than that of the labeling substance-containing member (b) (hereinafter, a labeling substance elution accelerating member). (Referred to as “(i)”).

2. The method according to claim 1, wherein the labeling substance elution promoting member (i) has an average flow pore diameter (mean flow pore size) of 10 im or more.

3. The method according to claim 2, wherein the labeling substance elution-promoting member (i) has an average flow pore diameter (mean flow pore size) of 30 to 50 zm.

4. The method according to claim 1, wherein the labeling substance elution promoting member (i) and the labeling substance-containing member (b) are made of the same material.

5. The method according to claim 4, wherein the labeling substance elution promoting member (i) and the labeling substance-containing member (b) are porosilicate bars.

6. The method according to claim 1, wherein the labeling substance-containing member (b) is sandwiched from above and below by the labeling substance elution promoting member (i).

7. The method according to claim 1, wherein the analyte is a complete antigen or a hapten.

8. The method according to claim 7, wherein the intact antigen to be analyzed is hCG.

9. In a simple immunochemical assay using hapten as the analysis target, (1) the hapten which is the analyte in the sample, and (2) the labeled analyte or a chemically modified product thereof, Antibody-to-antibody reaction with an analyte that is immobilized on the same chromatographic material and that is present on the same chromatographic material at different concentrations at different concentrations in order from the upstream to the downstream of the chromatographic material. 2. The method according to claim 1, wherein the concentration of the analyte in the sample is determined using the label as an index.

10. The method according to claim 9, wherein the concentration of the antibody against the analyte immobilized on the chromatographic material increases gradually from upstream to downstream of the chromatographic material.

11. The method according to claim 9, wherein the analyte hapten is estrogen or pregnanediol.

12. The sample addition section (A) for adding the sample containing the analyte, the specific binding substance to the labeled analyte, or the labeled analyte or its chemically denatured substance (labeled substance) Labeled substance-existing part (B) existing in a state where it can be chromatographed, one or more detection parts in which a specific binding substance to the analyte is immobilized

In a simple immunochemical qualitative or quantitative (semi-quantitative) analyzer using a chromatographic method including (C) and an added sample and an absorbing section (D) for absorbing and removing the labeling substance not bound to the detecting section (C);

A labeling substance elution promoting member (i) made of a material having an eye roughness equal to or greater than that of the labeling substance containing member (b) is laminated on the labeling substance containing member (b) constituting the labeling substance existing part (B). And a member (a) constituting the sample addition section (A) is brought into contact with the labeling substance elution accelerating member (i).

13. The apparatus according to claim 12, wherein the labeling substance elution accelerating member (i) has an average pore diameter of 10 / m or more.

14. The apparatus according to claim 13, wherein the labeling substance elution promoting member (i) has an average pore diameter of 30 to 50 m.

15. The apparatus according to claim 12, wherein the labeling substance-containing member (b) and the labeling substance elution promoting member (i) are made of the same material.

16. The material according to claim 15, wherein the material of the labeling substance-containing member (b) and the labeling substance elution promoting member (i) is borosilicate glass fiber.

17. The apparatus according to claim 12, wherein the labeling substance-containing member (b) is sandwiched from above and below by the labeling substance elution promoting member (i).

18. The apparatus according to claim 12, wherein the analyte is a hapten, and has a plurality of detectors (C! Cn) on a single attrib.

19. The apparatus according to claim 18, wherein the concentration of the hapten antibody in the plurality of detection sections (CiCr) increases sequentially from upstream to downstream of the chromatographic material.