WO1996024845A1 - Reagent for detecting substances and method for diagnosing rheumatoid arthritis - Google Patents

Abstract

Rheumatoid arthritis, etc., can be reliably, conveniently and quickly diagnosed by a method comprising: the step of reacting a membrane having one of galactose-deficient IgG and Ricinnus communis agglutinin I immobilized thereon, colored fine particles having the other of them immobilized thereon, and a biological sample taken up from a subject; and the step of detecting these colored fine particles captured on the membrane via the bond between the antigalactose-deficient IgG antibody contained in the sample and galactose-deficient IgG and the bond between Ricinnus communis agglutinin I and the galactose contained in the antigalactose-deficient IgG antibody.

Description

 Description Substance detection reagent and method for detecting rheumatoid arthritis

The present invention relates to a detection reagent for detecting a specific substance to be detected, in particular, an anti-galactose-deficient IgG antibody (an antibody against IgG lacking galactose) found in patients with rheumatoid arthritis; Diagnostic agents and methods for detecting rheumatoid arthritis.

Analysis of biological substances as a means of making bell-specific diagnoses of disease has been widely adopted in clinical tests. Depending on the purpose of the clinical test, a high-sensitivity and high-precision quantitative method and a qualitative method that is easy to operate and can obtain a result in a short time are appropriately used. As one of such methods, a method utilizing an immunological antigen-antibody reaction is widely used.

 At present, simple qualitative methods using immunological antigen-antibody reactions include enzyme immunoassay using an enzyme as a label (hereinafter sometimes abbreviated as EIA), particle agglutination using microparticles, agglutination inhibition, and A coloring method using colored fine particles is known. Among the coloring methods, the flow-through method and the immunochromatographic method (hereinafter sometimes referred to as the immunochromatographic method) are the most commonly known, and many diagnostic reagents utilizing these methods are commercially available. It has been used in clinical tests. Hereinafter, both methods will be described.

In the flow-through method, the target substance is specifically bound to the reactive substance-immobilized membrane in which the reactive substance 1 (antibody 1) that specifically binds to the target substance is immobilized on a part of the membrane and to the colored fine particles. This is a method using reactive fine particles on which reactive substance 2 (antibody 2) is immobilized. In the flow-through method, the specimen containing the substance to be detected and the reactive fine particles are filtered through a reactive substance-immobilized membrane. A complex sandwiched in a sandwich is formed. At this time, the color tone of the fine particles is recognized on the film, and the presence of the substance to be detected can be confirmed with the naked eye. As a typical method of the flow through method, a method described in Japanese Patent Application Laid-Open No. 63-127160 is known. The following is a specific description of a commercially available urinary human luteinizing hormone (hereinafter abbreviated as LH) detection kit (manufactured by Futatsubon Gene) as an example. Although this method is sometimes called the alias filter method, here it is called the flow-through method.

 First, an anti-LH monoclonal antibody 1 is applied to a dinitrocellulose membrane to prepare an antibody-immobilized membrane. On the other hand, anti-LH monoclonal antibody 2 is immobilized on gold colloid particles having a purple-red color tone, dispensed into vials one by one for each use, and lyophilized to prepare a gold colloid reagent for detection. When a predetermined amount of urine containing LH is added to the vial, the gold colloid reagent for detection is restored to a solution state by urine, and the (LH) -one (anti-LH monoclonal antibody 2)-(colloidal gold) complex is formed. It is formed. (LH)-(anti-LH monoclonal antibody 2)-(gold colloid particles) in the vial bottle When the complex solution was dropped into the antibody immobilization medium, the solution dropped while passing through the membrane in the thickness direction. (Membrane) One (anti-LH monoclonal antibody 1) one (LH) — (anti-LH monoclonal antibody 2) — (colloidal gold particles) complex that sandwiches LH in a sandwich, with red on the membrane Purple coloring can be visually confirmed. When urine does not contain LH, no sandwich-like complex is formed and no color appears. The time required for the determination in two steps is about 2 minutes, and urinary LH can be detected easily and quickly.

 As described above, the flow-through method using colored fine particles is a method in which the operation time is short, the operation is simple, and the judgment is easy.

The immunochromatography method is a method using a reactive substance-immobilized membrane and reaction fine particles as in the flow-through method. However, in the immunochromatography method, the specimen containing the substance to be detected and the reactive fine particles spread on the reactive substance-immobilized membrane by the same phenomenon as the paper chromatography method, and spread to the reactive substance-immobilized site on the membrane. When the flow arrives, Reactive Substance 1 (Antibody 1) and Reactive Substance 2 (Antibody 2) form a complex sandwiching the substance to be detected, and the color tone of the fine particles is observed on the membrane. Substance to be detected Is based on the principle that the presence of can be confirmed with the naked eye. The following is a specific description of a commercially available pregnancy test drug Clear Blue One Step-I S (manufactured by Unipass).

 The nitrocellulose membrane is cut into strips, and an anti-human chorionic gonadotropin (hereinafter sometimes abbreviated as hCG) monoclonal antibody 1 is immobilized near one end to form a reaction site. Next, the dried anti-hCG monoclonal antibody 2 immobilized blue latex particles in a dry state are held near the end opposite to the reaction site in a form movable by a developing flow. A urine sample containing hCG is dropped directly onto the absorption pad that has been brought into contact with the end holding the blue latex particles, and the urine sample is transferred from the absorption pad to the membrane, and further developed on the surface. It reaches the blue latex particle holding part. At this time, the hCG in the developing stream binds to the anti-hCG monoclonal antibody 2 on the blue latex particles to form the (hCG)-(anti-hCG monoclonal antibody 2) — (blue latex particle) complex . This complex continues to expand further, reaches the reaction site, and the hCG in the complex reacts with the anti-hCG monoclonal antibody 1, (membrane)-(anti-hCG monoclonal antibody 1) one (hCG) -(Anti hCG monoclonal antibody 2)-(Blue latex particles) forms a complex. At this time, strong blue coloring can be visually confirmed at the reaction site on the film. When urine does not contain hCG, no intense coloration appears since no sandwich-like complex is formed at the reaction site. The operation is a single step of simply dropping urine onto the absorption pad, and the time required for the determination is about 3 minutes, which is simple and quick. Thus, the immunochromatography method using colored fine particles has a short operation time, is easy to operate, and is easy to judge.

 As described above, the flow-through method and the immunochromatography method are widely used as diagnostic reagents. These diagnostic reagents are diagnostic reagents that use only the antigen-antibody combination. Almost no diagnostic reagents are known, and in particular, there are no diagnostic reagents by immunochromatography or flow-through using ricinascominisagglutinin I (RCA120).

By the way, rheumatoid arthritis (hereinafter sometimes abbreviated as RA) is one of the diseases that have attracted attention recently. An autoimmune antibody called rheumatoid factor (hereinafter sometimes abbreviated as RF) is present in the serum of RA patients. It is known to recognize the Fc site of Roblin G (hereinafter sometimes abbreviated as IgG) as an antigen. Current clinical chemistry diagnostic methods for RA include particle agglutination using animal IgG such as egrets, denatured human IgG, or microparticles having these Fc sites immobilized as antigens. For example, RA test (RA diagnostic method for qualitative detection of RF based on the presence or absence of aggregation of latex particles on a slide plate), RAPA method (abbreviation of RA-particle agglutination: aggregation of latex particles in a microtiter plate) , Sedimentation, semi-quantitative determination of RF) and TIA method (abbreviation of Turbid metric i band unoassay: quantitative method of RF for spectroscopically measuring the degree of aggregation of latex particles). In addition, a method of using a rheumatoid factor detected by an EIA method using an antigen immobilized on a microplate well as a disease marker for RA has also been adopted.

 In the current diagnosis of RA by the detection of rheumatoid factor using the particle agglutination method and the EIA method, a positive result may be obtained even when the serum of a healthy person is used as a sample. The sera of patients with rheumatoid diseases such as osteoarthritis are positive at a high rate, and the clinical specificity is poor. In addition, the positive rate of these methods in true RA patients (hereinafter sometimes referred to as clinical sensitivity) is not very high. Thus, it is widely recognized that conventional diagnosis of RA by detection of rheumatoid factor has low clinical sensitivity and clinical specificity in clinical practice. However, since there is no clinical chemistry method other than these that can be used for the diagnosis of RA, it is currently used widely.

 In addition to the problems described above, the particle agglomeration method requires a simple operation, and the time required to obtain a result is about 3 minutes to 2 hours.However, the judgment involves the subjectivity of the tester. To perform an accurate determination, the skill of the inspector is essential.

In the EIA method, the operation is complicated, requires about 2 to 4 hours of operation time, and requires specialized equipment such as a spectrophotometer and a microplate reader for the determination.

Recently, detailed analysis was performed on the sugar chains present at the IgG Fc site in the serum of RA patients, and as a result, the sugar chains were found to be located at the IgG Fc site in the serum of healthy individuals. It is reported that the galactose content is significantly reduced compared to the sugar chains present. [Nature, Vol. 316, pp. 452, 1985]. That is, the sugar moiety present at the Fc site of IgG in the serum of a healthy human is composed of a plurality of sugar chains having different structures, and the abundance ratio between the types of sugar chains is almost constant among individuals. It was made clear. On the other hand, the sugar moiety at the Fc site of IgG in the serum of RA patients is composed of multiple types of sugar chains with different structures. As in the above, it was found that the content of galactose was almost constant among individuals, or that the galactose content was significantly reduced as a whole. More specifically, three types of sugar chains containing two, one, and zero galactose molecules, respectively, are contained in the sugar portion of the Fc site of IgG in healthy human serum at about 2: 2: 1. However, in the glycan of the Fc site of IgG in the serum of RA patients, the type of glycans containing two galactose molecules is significantly reduced, and the total number of glycans deficient in galactose is significantly increased. It is reported that it is increasing. Therefore, based on this fact, the sugar moiety at the Fc site of IgG in the serum of RA patients has a sugar chain structural abnormality, and if this structural abnormality can be identified, it will It was expected to be a powerful marker.

 For this reason, several diagnostic methods that utilize structural abnormalities in the sugar chain at the Fc site of I.gG in the serum of RA patients have been proposed as alternatives to the conventional methods. These methods are broadly divided into two types according to the detection target. One is to take advantage of the fact that the galactose content in the sugar chain at the IgG Fc site in the serum of RA patients decreases, and to reduce the galactose content in the sugar chain at the IgG Fc site in the serum. A method of measuring and comparing with that of a healthy person, and diagnosing RA when the galactose content is lower than that of a healthy person (Japanese Patent Application Laid-Open (JP-A) Nos. 3-73857 and 5-87874) Gazette). The other is that autoantibodies against structural abnormalities in the sugar chain at the Fc site of IgG, that is, anti-galactose-deficient IgG antibodies are present in the serum of RA patients. This is a method for preparing an anti-galactose-deficient IgG antibody in serum against this (JP-A-3-48070).

Among these two methods, the method of measuring the galactose content in the sugar chain of IgG in serum is very complicated and requires a long reaction time of several hours. In the method of No. 814, the total amount of IgG in the serum must be measured simultaneously for each sample and a scintillation counter must be used, and the solution of complex data before determination This method has the disadvantage of requiring analysis, and the clinical sensitivity and clinical specificity of the determination have not been satisfactory.

 On the other hand, the method of measuring an anti-galactose-deficient IgG antibody involves quantifying the anti-galactose-deficient IgG antibody using a method similar to EIA, and the clinical sensitivity and clinical specificity of the determination are low. It is very good and can make a reliable diagnosis of RA. However, since this method is a quantitative method, the operation is complicated, it takes 4 hours to overnight to obtain a result, and it has the drawback that instruments such as a spectrophotometer and a densitometer must be used. Was. DISCLOSURE OF THE INVENTION The present invention has been made from the above viewpoints, and is a diagnostic reagent and method for accurately, simply, and quickly diagnosing rheumatoid arthritis, and a detection reagent for detecting other substances to be detected. It is another object of the present invention to provide a detection reagent to which conventional immunochromatography and flow-through methods can be applied.

 The present inventors have conducted intensive studies in order to solve the above problems, and as a result, a detection reagent used in a detection method such as an immunochromatography method and a flow-through method using ricinus minisagglutinin I (RCA120). The present invention was completed by applying this method to a detection reagent for easily detecting an anti-galactose-deficient IgG antibody in a human body fluid and a diagnostic reagent for rheumatoid arthritis.

 That is, the present invention is a detection reagent for detecting a substance to be detected having a sugar chain containing galactose and / or yS-N-acetylgalactosamine at the terminal, wherein the first reactive substance is immobilized. One of the reactive substances is lisinascominisagglutinin I, and the other of the reactive substances specifically binds to the substance to be detected. This is a detection reagent that is a reactive substance having no sugar chain and containing galactose and ^ -N-acetylgalactosamine at its terminals.

The present invention also relates to a detection reagent for detecting an anti-galactose-deficient IgG antibody, wherein the detection reagent comprises a membrane and fine particles, and one of the membrane and the fine particles has immobilized lisinasukominisagglutinin I; On the other hand, galactose-deficient IgG was immobilized. Provided detection reagents.

 The present invention further provides a diagnostic agent for rheumatoid arthritis comprising the detection reagent; a membrane on which one of galactose-deficient IgG and ricinosaminisagglutinin I is immobilized; and a colored microparticle on which the other is immobilized. Reacting with a biological sample obtained from the subject;

 Through the binding between the anti-galactose-deficient IgG antibody and the galactose-deficient IgG contained in the sample, and the binding between ricinus-cominisagglutinin I and the galactose contained in the anti-galactose-deficient IgG antibody, Detecting colored particles trapped on the membrane.

Provide a method for detecting rheumatoid arthritis.

 Hereinafter, the present invention will be described in detail.

<1> Detection reagent for detecting the target substance

 The detection reagent for detecting a target substance of the present invention is a detection reagent for detecting a target substance having a sugar chain containing terminal galactose and / or e-N-acetylgalactosamine. A membrane on which the first reactive substance is immobilized, and fine particles on which the second reactive substance is immobilized. One of these reactive substances is ricinus comminisagglutinin I, and the other of the reactive substances specifically binds to the substance to be detected and has a sugar chain at the end containing galactose and -N-acetylgalactosamine. Not a reactive substance.

 The above-mentioned detection reagent utilizes the specific binding of galactose or 8-N-acetylgalactosamine and ricinosaminus glutglutinin I, which specifically binds thereto, to the flow-through method and the immunochromatography method. The binding between the analyte having a sugar chain containing galactose or -N-acetylgalactosamine contained in the sample and ricinosaminisagglutinin I, and the specific binding between the analyte and the analyte The detection of the colored fine particles captured on the membrane through the binding with a reactive substance having no sugar chain containing galactose and 9-N-acetylgalactosamine binds to the substance to be detected qualitatively. Is to be detected.

Hereinafter, the detection reagent of the present invention will be described in detail. (1) Fine particles

 The fine particles are a marker for visually judging the presence of the substance to be detected, and are preferably colored in order to facilitate the naked eye judgment. Examples of the material include water-insoluble materials such as uniform spherical particles composed of a synthetic polymer such as polystyrene latex or natural polymers such as gelatin, and gold-colloid particles such as gold colloid. The metal colloid particles do not need to be colored because of their inherent colors. What is colorless may be appropriately colored using a dye. The particle size of the fine particles must be smaller than the pore size of the membrane, but can be used generally in the range of 0.01 to 5 m, and particularly preferably in the range of 0.05 to 2 m.

(2) membrane

 The membrane used in the present invention is for indirectly capturing the above-mentioned fine particles through the binding between the substance to be detected and ricinascominisagglutinin I, and can immobilize the reactive substance. Further, any unreacted fine particles can be passed in the flow-through method, and any fine particles capable of developing the fine particles in an aqueous solution as a chromatographic carrier can be used in the immunochromatographic method. Examples of the material include a porous three-dimensional structure membrane, for example, a nylon membrane and a nitrocellulose membrane, and may be either a synthetic or natural polymer membrane. The pore size of the membrane may be any size as long as the reaction fine particles can pass through without causing clogging, but it is particularly preferably in the range of 0.2 to 8 / m.

(3) First reactive substance and second reactive substance

One of the first reactive substance and the second reactive substance is ricinascominisagglutinin I (hereinafter referred to as “RCA 120”). RCA120 is a lectin derived from chickpea (Ricinus co madison unis) having an activity of specifically binding to galactose and -N-acetylgalactosamine. JU Baenziger) et al. (The 'Journal of Biological Chemistry, 254, 9795-9799, 1979) by purifying from bean paste. Also, commercially available products may be used. In addition, galactose other than RCA12 0 or) 3 N Lectins that specifically bind to cetyl galactosamine, such as ricinosaminisglutinin II (RCA 60), beetle sialglutinin (Alio A), pine shroom lectin (ABA), peanut lectin (PNA), and the like, are also described in the present invention. It can be used for Of these, RC A 120 is particularly preferred.

 The other of the first reactive substance and the second reactive substance is a substance capable of detecting the target substance by combining with RCA120. It is necessary that this reactive substance does not bind to RCA120, and therefore does not need to have a sugar chain containing galactose or 1N-acetylgalactosamine at the end. Hereinafter, this reactive substance is referred to as “galactose-deficient reactive substance”. Specific examples thereof include galactose-deficient IgG, galactose and a galactose-deficient antibody against a glycoprotein containing a sugar chain containing) 3-N-acetylgalactosamine.

(4) Immobilization of each reactive substance on fine particles and membrane

 The method of fixing the first and second reactive substances to the fine particles and the membrane may be a physical adsorption method, a chemical bonding method, or any other method. That is, any immobilizing means may be used as long as the reactive substance such as RCA 120 does not desorb from the fine particles and the membrane.

 In the following description, the membrane on which the reactive substance is immobilized is referred to as “reactive substance-immobilized membrane”, and the fine particles on which the reactive substance is immobilized are referred to as “reactive microparticles”.

(5) Substance to be detected

 The substance detected by the detection reagent of the present invention is a substance having a sugar chain containing galactose and / or ^ -N-acetylgalactosamine at its terminal, and specifically binding to RCA120. . Specifically, it is an anti-galactose-deficient IgG antibody, a glycoprotein having a sugar chain containing galactose and / or / SN-acetylgalactosamine at its terminal. Examples of such glycoproteins include thyroglobulin, transfurin and the like.

When the substance to be detected is an anti-galactose deficient IgG antibody, the galactose deficient reactive substance is galactose deficient IgG. Is a galactose-deficient antibody against a glycoprotein.

(6) Detection reagent and its use

 Hereinafter, detection reagents using the above materials and methods for using the same will be described. In the following description, an example using a flow-through method will be described for more specificity, but the present detection reagent can also be carried out by an immunochromatography method. When the immunochromatography method is carried out, it can be carried out by adapting the shape of the membrane, the site where the reactive substance is immobilized to the membrane to the immunochromatography method, and keeping the reactive fine particles on the membrane in a dry state.

 The detection reagent includes a membrane on which the first reactive substance is immobilized, fine particles on which the second reactive substance is immobilized, and, if necessary, a reaction container, a solution for diluting a sample, and a washing solution. .

 The reactive microparticles obtained as described above contain about 0.05 to 3% (w / w) of serum albumin to maintain the chemical stability of colloid and the specific binding activity of the reactive substance. It is suspended in a buffer solution containing about 0.05 to 10% (w / w) of a water-soluble polymer such as polyethylene glycol, other stabilizers, and a preservative, and stored in a cool place. In addition, the suspension can be freeze-dried for long-term storage, and dissolved in distilled water at the time of inspection for use.

 On the other hand, the reactive substance-immobilized membrane is subjected to a blocking treatment with about 0.05 to 5% (w / w) of serum albumin to prevent nonspecific adsorption of the specimen or the reactive fine particles. And store under dry conditions.

(Reaction vessel)

 When the detection reagent of the present invention is used by a flow-through method, it is preferable that the flow rate and the flow rate of the reagent involved in the reaction and the specimen are stable and easy to use. Therefore, a reaction container is required, but the reaction container is made of plastic or other material, and a window is provided for dropping each reagent and sample into the container.A molded container, an absorbent, and a reactive substance It is composed of an immobilized membrane and other necessary members.

It is desirable that the absorbent material be capable of sufficiently absorbing each drop reagent used in the test, and have a performance that does not fluctuate the absorption rate of each dropped reagent and sample. like that As the material, cotton, non-woven fabric, base paper, porous plastic and the like are preferable, but any material having the above-mentioned performance may be used. The size of the absorbent may be any size as long as the reagents and specimens dropped during the test are completely absorbed.

 In the reaction vessel, the reactive substance-immobilized membrane is placed on the absorbent in the vessel, and these are fixed to the vessel in order to prevent movement in the vessel. At this time, adjust the position where the reactive substance-immobilized membrane and the absorbent are fixed to the window for dropping each reaction reagent and sample. A tissue, a mesh, etc., to complete these contacts, and a paper, etc., to adjust the absorption rate of each reagent and sample may be interposed between the absorbent and the reactive substance-immobilized membrane. .

[Other reagents (sample dilution solution and washing solution)]

 When performing detection with the reagent of the present invention, the sample may be used directly without dilution, but when performing semi-quantitative testing, and the material and production of the reactive substance-immobilized membrane and reactive fine particles Samples should be diluted as needed according to method and nature. In this case, as a sample diluting solution, physiological saline, various buffers or these solutions were added with about 0.05 to 3% (w / w) of serum albumin or a surfactant. A liquid can be used as appropriate. In the flow-through method to which the present invention is applied, if a washing solution to be used after dropping the sample and the reaction fine particle suspension is also required, a physiological saline solution, various buffer solutions, or a solution containing 0.05 to 3 of these solutions may be used. A solution containing about% (w / w) of serum albumin or a surfactant can be used as appropriate.

(Detection kit)

A detection kit containing necessary solutions and reagents in one package so that the user can detect the substance to be detected of the present invention without using special equipment or preparing solutions or reagents. It can also be. Specifically, in one package, the instructions for use, the reaction microparticle suspension, the reaction vessel incorporating the reactive substance-immobilized membrane, and the sample diluent and washing solution as needed are combined for the desired number of measurements. And use it as the detection kit. 〔how to use〕

 As the detection reagent of the present invention, as described above, a reaction microparticle suspension in which RCA 120 is immobilized, a reaction vessel incorporating a reactive substance-immobilized membrane in which a galactose-deficient reactive substance is immobilized, The use of the detection kit by the flow-through method including the sample diluting solution and the washing solution will be described below.

 A sample (either directly or diluted with a sample diluting solution) is dropped on the reactive substance-immobilized membrane through the window of the reaction vessel, and is completely absorbed by the absorbent material. The suspension is dropped onto the reactive substance-immobilized membrane, absorbed by an absorbent, and visually observed for coloring on the membrane. If the analyte contains a sugar chain containing galactose and Z or ^ -I-N-acetylgalactosamine and has a substance to be detected that specifically binds to a galactose-deficient reactive substance, the galactose immobilized on the membrane The target substance binds to the deficient reactive substance, and RCA120 binds to galactose or ^ -N-acetylgalactosamine contained in the target substance. As a result, fine particles are captured on the membrane.

 Therefore, if the reaction microparticles with RCA120 immobilized on the membrane are colored, the analyte is present in the sample and determined to be positive, and if not, the analyte is detected. Does not exist, that is, it can be determined to be negative. In the case of no coloration in the competitive reaction, it may be judged as positive. The process from the dropping of the sample to the determination of the presence or absence of coloring can be performed in 30 seconds to 10 minutes. The operation is a two-step operation. The washing liquid may be dropped after dropping the sample and after dropping the reaction fine particles. In this case, there are three or four steps. In addition, the film after the determination can be dried and stored for a long time without a change in coloring.

In the present invention, the flow-through method is a method of physically separating BZF separation (bound form: B und, B formed by binding of an antibody in an antigen-antibody reaction and free form: Free, F not bound). This is a particularly effective method for detection systems that require However, in a detection system that does not require B / F separation, that is, a system in which there is substantially no contaminant that affects the specific binding between RCA120 and the target substance, the reaction fine particles are in a dry state. It can be carried out as a one-stage Atsey method, in which the fine particles are fixed to the upper part of the membrane by a sample dropping operation and the reaction fine particles are returned to a solution state by a sample dropping operation. Also, cut the membrane into strips, An immunochromatography method using a test paper in which a reactive substance is immobilized on one end and reactive fine particles are held in a dry state on the other end can also be performed.

<2> Detection reagent for anti-galactose-deficient IgG antibody

 The reagent for detecting an anti-galactose-deficient IgG antibody of the present invention comprises a membrane and fine particles, and one of the membrane and the fine particles has RCA120 immobilized thereon, and the other has galactose-deficient IgG immobilized thereon. I have.

 The membrane, microparticles, RCA120, reactive microparticles, reactive substance-immobilized membrane, reaction vessel, specimen diluting solution, washing solution, and the like are the same as those described in <1> above. The present detection reagent is characterized in that the substance to be detected is an anti-galactose-deficient IgG antibody, and one of the reactive substances is galactose-deficient IgG.

 The anti-galactose-deficient IgG antibody detection reagent and the detection method using the same according to the present invention will be described using a flow-through method as an example. A blood sample, which is a normal sample, requires BZF separation due to the presence of a large amount of immunoglobulin, glycoproteins, polysaccharides, etc. other than anti-galactose-deficient IgG antibodies, so that the detection reagent of the present invention was used. Therefore, it is preferable to use a flow-through method. The preparation method of each reaction reagent is in accordance with <1>.

CRCA 120 immobilized particulate suspension)

 RC A 120 as a reactive substance is immobilized on colored microparticles to obtain an RCA 120 immobilized microparticle suspension.

(Galactose-deficient IgG immobilized membrane)

The reactive substance is defined as galactose-deficient IgG, and a galactose-deficient IgG-immobilized membrane is prepared. Galactose-deficient IgG is obtained by subjecting human IgG to degalactosylation using an enzyme according to a known method (see JP-A-3-48700 or JP-A-5-87814), and purifying it. Obtain a IgG lacking toose. Confirm that galactose is almost completely removed by ion-exchange chromatography. Next, this is fixed to a membrane such as a membrane filter by means of spotting, spraying or printing to prepare a galactose-deficient IgG immobilized membrane. The amount to be fixed is the final detection Although it can be appropriately adjusted depending on the sensitivity of the reagent and the like, a value between 0.5 and 10 g is particularly desirable.

(Reaction vessel)

 An absorbent and a galactose-deficient IgG immobilized membrane are incorporated in a plastic container in the same manner as above to prepare a reaction container.

(Anti-galactose-deficient IgG antibody detection kit)

 In order for the user to detect the anti-galactose-deficient IgG antibody of the present invention without using special equipment, preparing solutions and reagents, incorporate necessary solutions and reagents in one package. May be used as a detection kit. Specifically, in one package, instructions for use, the RCA120-immobilized fine particle suspension, the galactose-deficient IgG-immobilized membrane-incorporated reaction vessel, and the sample diluent and Combine the washing solution with the desired number of measurements and use it as an anti-galactose-deficient IgG antibody detection kit.

[Using anti-galactose-deficient IgG antibody detection reagent]

 The anti-galactose-deficient IgG antibody is detected using the above-described anti-galactose-deficient IgG antibody detection reagent as follows.

 A sample (either directly or diluted with a sample diluent) is dropped onto the galactose-deficient IgG immobilized membrane through the window of the reaction vessel, allowed to be completely absorbed by the absorbent material, and then the RCA120 immobilized fine particles are removed. The suspension is dropped onto a galactose-deficient IgG immobilized membrane, absorbed by an absorbent, and visually observed for coloration on the membrane. Anti-galactose deficiency is detected when RCA 12 0-immobilized microparticles are stained 1 g G antibody is present in the sample and determined to be positive.If no staining is observed, anti-galactose deficiency is detected. It is determined that no IgG antibody is present, that is, negative. The samples (biological samples) used in the anti-galactose-deficient〗 gG antibody detection reagent include blood (whole blood), serum, plasma, saliva, synovial fluid, and the like.

(Reaction principle)

The reaction principle in the use of the anti-galactose-deficient IgG antibody detection reagent is shown below based on FIG. In Figure 1, the anti-galactose-deficient IgG antibody was Although the antibody is shown for convenience, the antibody is not limited to the IgG type, and may be another type of antibody.

 In the first reaction, the sample is dropped on the galactose-deficient IgG-immobilized membrane, and the anti-galactose-deficient IgG antibody in the sample reacts with the galactose-deficient IgG on the membrane, causing an antigen-antibody reaction. (Membrane) 1 (Galactose deficient IgG) — (Anti-galactose deficient IgG antibody) forms a complex.

 In the second reaction, when the RCA120-immobilized fine particles are dropped onto the membrane, the (membrane) one (galactose-deficient IgG) — (anti-galactose-deficient IgG antibody) anti-galactose-deficient in the complex RCA120 binds to galactose in the sugar chain present in the IgG antibody molecule, and (membrane)-(galactose-deficient IgG)-(galactose in anti-galactose-deficient IgG antibody) One (RCA120-immobilized fine particle) complex is formed, and the color of the fine particles can be visually confirmed as apparent coloring on the membrane. When the substance to be detected is an anti-galactose-deficient IgG antibody in the serum of a patient with rheumatoid arthritis, particularly an IgG-type antibody, most of the antibodies lack galactose. However, all IgG-type antibodies are not completely deficient in galactose but some are still present, so they can bind to RCA120.

 When no anti-galactose-deficient IgG antibody is present in the sample, RCA120 does not bind to galactose-deficient IgG, and no complex is formed in the first reaction. Passes through the membrane and no coloring is observed. Also, based on the principle of this method, the intensity of coloring varies depending on the amount of anti-galactose-deficient IgG antibody in the sample, so the coloring intensity is measured with a densitometer, colorimeter, etc. This makes it possible to quantify the amount of anti-galactose-deficient IgG antibody in the sample. As described above, an anti-galactose-deficient IgG antibody detection reagent is provided. This reagent can be used to qualitatively or easily detect anti-galactose-deficient IgG antibodies in a sample in a short time of 0.5 to 10 minutes and in a simple operation of 2 to 4 steps. It can be detected semi-quantitatively. <3> Diagnostic reagent for rheumatoid arthritis (RA)

The anti-galactose-deficient 1 gG antibody detection reagent according to the present invention comprises, as described above, The anti-galactose-deficient IgG antibody in it can be reliably detected. By the way, Japanese Patent Application Laid-Open No. Hei 3-4870 discloses that a method for quantifying an anti-galactose-deficient IgG antibody enables accurate diagnosis of rheumatoid arthritis. I have. From these facts, if the anti-galactose-deficient IgG antibody detection reagent according to the present invention is applied to rheumatoid arthritis as a diagnostic object, it can be used in the same manner as the quantitative method of the invention described in Japanese Patent Application Laid-Open No. 3-470000. Accurate diagnosis of rheumatoid arthritis is possible.

 Therefore, the reagent for detecting an anti-galactose-deficient IgG antibody according to the present invention can be used as a diagnostic reagent for rheumatoid arthritis (RA).

 If this anti-galactose-deficient IgG antibody detection reagent is used in a clinical test as a diagnostic reagent for rheumatoid arthritis (RA), the method of quantification according to the invention described in Japanese Patent Application Laid-Open No. 3-4870000 can be used in addition to the conventional aggregation method. It is possible to accurately, simply and quickly diagnose rheumatoid arthritis, and is useful as a diagnostic method for rheumatoid arthritis.

<4> Detection of rheumatoid arthritis

 Using the diagnostic agent for rheumatoid arthritis, rheumatoid arthritis can be detected. That is, the method for detecting rheumatoid arthritis of the present invention comprises:

 A step of reacting a membrane in which one of galactose-deficient IgG and RCA120 is immobilized, a colored microparticle in which the other is immobilized, and a biological sample collected from a subject;

 Through the binding between the anti-galactose-deficient IgG antibody contained in the sample and the galactose-deficient IgG antibody and through the binding between ricinus comminus agglutinin I and the galactose contained in the anti-galactose-deficient IgG antibody And detecting a colored fine particle captured on the film. Next, the present invention will be described in detail with reference to test examples.

 (Test Example 1)

In this test, the detection method using the detection reagent of the present invention (this method) and the quantification method described in Examples 1 to 3 of the invention (conventional method) described in Japanese Patent Application Laid-Open No. 3-470000 are used. Compare Went for.

(1) Sample specimen

 As a sample specimen, the above-mentioned standard serum of a patient with rheumatoid arthritis (RA) [Rheumatoid Factor Control / Calibrator: manufactured by INTERNATIONA L ENZYMES, INC. Catalog No. 71 1 2, 7 1 1 3, 7 1 1 4 A total of 17 specimens were used: 6 specimens, 8 specimens of patients with rheumatoid arthritis (RA), and 8 specimens of serum from healthy subjects.

(2) Test method

 1) Detection method using the detection reagent of the present invention (hereinafter referred to as the present method)

 An anti-galactose-deficient IgG antibody detection reagent was prepared according to Example 1 described below, and qualitatively detected the anti-galactose-deficient IgG antibody using the reagent.The presence or absence of red spots observed in the reaction container window was confirmed. Was determined to be rheumatoid arthritis (RA) positive or negative.

2) Conventional method

 The anti-galactose-deficient IgG antibody in the sample was quantified according to a method similar to EIA.

The operation of the method similar to EIA used a plastic microtiter well in which galactose-deficient IgG was immobilized and subjected to a blocking treatment in advance. Dilute the sample, add 1001 to the well, and incubate for 2 hours. After washing, add biotin-labeled RC A120 and incubate for an additional hour. After washing, avidin-labeled horse radish peroxidase was added and incubated for 1 hour. After washing, the substrate was reacted for 30 minutes to develop color. The color development concentration in the microtiter well was measured using a commercially available microplate reader (manufactured by BioRad). The value obtained by multiplying the absorbance by the dilution factor was used as the quantitative measurement value. The cutoff value of the value measured by the quantitative method was set to 100, and rheumatoid arthritis (RA) was judged negative for less than 100 and positive for 100 or more.

Using the 17 samples, each sample was tested by the present method of 1) and the conventional method of 2). For more information, this method will be used for rheumatoid arthritis (RA) positive or After judging negative for each sample, then, according to the conventional method, for each group that became rheumatoid arthritis (RA) positive or negative by this method, for each sample, rheumatoid arthritis (RA) positive Or negative was determined for each sample.

(3) Test results

 The results of this study are shown in Table 1, which is written by Robert H. Fletcher et al. (Translated by Shigeru Kumichi et al.), Epidemiology for clinical use. This is compiled with reference to Figure 3-4 on page 59 (Medical College, 1986). The group that was determined to be rheumatoid arthritis (RA) positive by this method is shown in the first row, and the number of specimens that were also determined to be positive by the conventional method is shown in column 1. The number of samples determined is shown in the second column. Similarly, the group that was determined to be rheumatoid arthritis (RA) negative by this method is shown in the second row, and the number of specimens that were determined to be positive by the conventional method is shown in the first column. The number of specimens judged to be negative is also shown in Table II. In addition, the sum of the results of the rheumatoid arthritis (RA) judgment by this method is summarized in Table 3 and the sum of the results of the rheumatoid arthritis (RA) judgment by the conventional method is summarized in the third row. Was.

 As is evident from Table 1, the results of comparison of the rheumatoid arthritis (RA) positive or negative judgment between the present method of 1) and the conventional method of 2) were compared. ) Positives were positive by the conventional method as they were, and those negative by the present method were negative by the conventional method as they were, and the concordance rate of the results was 100%.

In the conventional method 2), the operation was complicated and took 4 and a half hours. However, the number of operation steps of the anti-galactose-deficient IgG antibody detection reagent by the detection method using the detection reagent of the present invention (this method) was as follows. The operation is simple, with only two steps of dropping and dropping of the suspension of RCA120-immobilized fine particles, and the time required from dropping of the sample to determination is as fast as 2 minutes. The determination of chronic rheumatoid arthritis (RA) positive or negative using the galactose-deficient IgG antibody detection reagent was in good agreement with the conventional quantification method, indicating that the reagent of the present invention was used for the anti-galactose in human serum. Accurate toose-deficient 1 gG antibody It is clear that the detection is rapid. Therefore, it was found that the diagnosis of rheumatoid arthritis (RA) can be performed accurately, simply, and quickly.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram showing the reaction principle in the present invention.

 FIG. 2 is a plan view showing a detection reagent of one example of the present invention.

 FIG. 3 is a cross-sectional view of the detection reagent shown in FIG. 2 in the AA ′ plane. BEST MODE FOR CARRYING OUT THE INVENTION Next, the present invention will be described more specifically by showing examples. The present invention is not limited to the following examples. Example 1 Example of an anti-galactose-deficient IgG antibody detection reagent for flow-through method using RCA 120 and a detection method using the same <1> Preparation of galactose-deficient IgG

 It was prepared as follows by a known method (Japanese Unexamined Patent Publication No. 3-48070).

Human IgG protein 10 mg 1 in 0.1 M acid buffer (pH 5.0) The mixture was treated with sialidase (50 OmUZm 1), added with 0.1 M citrate monophosphate buffer (pH 7.0), and treated with / S-galactosidase (10 OmUZm 1). After these enzyme treatments, a 10-fold amount of glycine buffer (1.5 M glycine monohydrochloride, 3 M sodium chloride, pH 8.9.) To remove BSA, sialidase, β-peroxidase, etc. contained in the enzyme treatment solution using Protein C-Sepharose CL-4B to remove galactose deficiency from the enzyme treatment solution. IgG was purified. That is, the enzyme-treated sample was added to Protein C -Sepharose CL-4BC1 x 7.8CIB) which had been equilibrated with the binding buffer in advance, and the column was thoroughly washed with the binding buffer, and then 0.1 M glycine- Galactose-deficient IgG was recovered using hydrochloric acid (pH 3.0). At the time of recovery, in order to avoid leaving galactose-deficient I £ 〇 at a state of 13.0 for a long time, bind a galactose-deficient Ig to the same amount as the fractionation volume in advance. G was placed in a fraction tube for recovery. After collection, galactose-deficient IgG was sufficiently dialyzed against phosphate buffer (1 O mM phosphate, 0.15 M sodium chloride, pH 7.2).

<2> Preparation method of galactose-deficient IgG immobilized membrane

 1 g of the galactose-deficient IgG (5 mg / m 1) prepared in <1> above was spotted onto a nitrocellulose membrane (manufactured by Adobe Systems Incorporated) with a pore size of 3 μτη using a micropip. did. Dry thoroughly and then use blocking buffer (50 mM Tris-HCl, 0.15 M sodium chloride, 0.1% BSA (w / w), pH 8) to avoid non-specific adsorption. .0, hereinafter simply referred to as a blocking buffer) and dried sufficiently to obtain a galactose-deficient IgG-immobilized membrane.

<3> Preparation method of R C A 120 immobilized fine particles

RCA120 (2.5 mg / m from Honen Corporation) was diluted to a concentration of lmg / ml with Tris buffer (50 mM Tris-HCl, pH 8.0). To 0.9 ml of this solution, 100 μl of red carboxylated latex having a particle size of 0.3 / m (solid content: 10%, manufactured by Nippon Synthetic Rubber Co., Ltd.) is added, and the mixture is added at 45 ° C. for 60 minutes. The mixture was stirred, centrifuged (8000 rpm for 30 minutes), 1 ml of the above Tris buffer was added to the precipitate to disperse the mixture, and the mixture was centrifuged again (8000 rpm for 30 minutes). Disperse it in Tris buffer (50 mM Tris-hydrochloric acid, pH 8.0) containing 1% 833, 0.15 M sodium chloride and 0.02% sodium azide, and suspend the RCA120-immobilized microparticle suspension. A suspension was obtained.

<4> Assembling the reaction vessel

 2.5 cm x 2.5 cm x 1.0 cm cube, cut into an appropriate size into a plastic container 1 with a circular window 2 with a diameter of 0.5 cm at the reagent dropping part Absorbent cotton 3 was incorporated, and 1 cm square cut paper No. 6 (manufactured by Advantech) (4) was placed thereon, and the galactose-deficient IgG cut into 1 cm square was fixed thereon. The membrane 5 was placed with the galactose-deficient IgG spotted on the window of the container and fixed to obtain a reaction container (Figs. 2 and 3).

<5> Assembly of anti-galactose-deficient IgG antibody detection reagent

 The anti-galactose-deficient IgG antibody detection reagent was obtained by combining the RCA120-immobilized fine particle suspension and the galactose-deficient IgG-immobilized membrane-incorporating reaction container.

<6> Implementation of detection method (flow-through method) using anti-galactose-deficient IgG antibody detection reagent

Normal serum and RA patient standard serum [Rheumatoid Factor Control / Calibrator: Catalog No. 7 1 1 2] from International ENZYMES, INC. Were collected in 1001 micropigs each. Each was dropped into the window of the reaction container of the anti-galactose-deficient IgG antibody detection reagent. Absorb the sample completely, and immediately drop 200 ^ 1 of RCA120-immobilized particulate suspension of anti-galactose-deficient IgG antibody detection reagent 200 ^ 1 into each reaction vessel, and immediately absorb the suspension. After the coloring, the presence or absence of coloring was visually confirmed. As a result, the color of the white nitrocellulose membrane was not changed at all in the reaction vessel window where the healthy human serum was dropped, and no coloring was observed. On the other hand, a red spot was clearly observed in the window of the reaction vessel into which the RA standard serum had been dropped, and was judged to be R & D. The operation was performed in two steps: dropping the sample and dropping the suspension of RCA120-immobilized microparticles, and was extremely simple. The time required from the dropping of the sample to the determination was 2 minutes, which was quick.

 As described above, the present invention provides a reagent for detecting an anti-galactose-deficient IgG antibody for a flow-through method using RCA120.

Example 2 Example of a detection reagent for thyroglobulin for immunochromatography using RCA 120 and a detection method using the same

<1> Method for preparing galactose-deficient anti-thyroglobulin monoclonal antibody Was. That is, 1 mg of an anti-thyroglobulin monoclonal antibody [Anti Thyroglobulin (Mono): manufactured by Biomeda] was treated with sialidase and / S-galactosidase to prepare a galactose-deficient anti-thyroglobulin monoclonal antibody.

<2> Preparation of gold colloid immobilized with galactose-deficient anti-thyroglobulin monoclonal antibody

A solution prepared by diluting gold colloid (Gold colloid, Era GC 10: manufactured by Biocell) with Tris buffer (50 mM Tris-HCl, pH 8.0) and adjusting the absorbance at 540 nm with an optical path length of 1 cm to 1.0. To 20 ml, 0.9 mg of the galactose-deficient anti-thyroglobulin monoclonal antibody prepared in <1> above was mixed, and the mixture was stirred at 45 ° C. for 60 minutes. This was centrifuged at 50,000 g for 20 minutes, the supernatant was carefully removed, and the reddish purple pellet-like sediment was dispersed in 20 ml of the Tris buffer. Further, this dispersion was centrifuged under the same conditions, and the precipitate was redispersed in 5 ml of the above-mentioned Tris buffer containing 0.1% BSA and 10% sucrose to immobilize a galactose-deficient anti-thyroglobulin monoclonal antibody. A gold colloid suspension was obtained. <3> Preparation of test strips

 A nitrocellulose membrane with a pore size of 3 / m (made by Advantech) is cut into strips of 0.5 cm width and 6 cm length, and RCA 120 (2.5 mg Zm) is placed at 1 cm from the edge of the strip. l, manufactured by Honen Corporation) was spotted at 1 ng using a micropip and air-dried. The strips were then immersed in blocking buffer to avoid the effects of non-specific adsorption. The aforementioned Tris buffer containing 30% sucrose was placed at a position 1 cm from the end opposite to the RCA 120 coated portion of the strip that had been sufficiently dried, and then dried and then dried. A suspension of galactose-deficient anti-thyroglobulin monoclonal antibody-immobilized gold colloid was placed on the sucrose-applied portion at 501 points and air-dried to obtain a test paper strip.

<4> Assembling the reagent for detection of thyroglobulin

 The celluloid plate was cut into a strip having a width of 0.5 cm and a length of 6 cm, and the test paper pieces were overlapped and bonded using an adhesive or a double-sided tape. Further, a base paper cut to a width of 0.5 cm and a length of 2 cm was adhered to the end of the test piece on the side where the gold colloid was applied as an absorption pad for holding a sample, thereby obtaining a thyroglobulin detection reagent. .

<5> Implementation of a detection method using a thyroglobulin detection reagent (immunochromatography method) A thyroglobulin solution prepared by diluting thyroglobulin (Thyroglobulin, Human: manufactured by Chemicon) to 1 jug / ml by diluting with the above tris buffer, Using the fur as a sample, the absorption pad of the thyroglobulin detection reagent was directly immersed in the sample to confirm that the sample had been sufficiently absorbed. Then, the thyroglobulin detection reagent was kept horizontal and allowed to stand. When the sample begins to develop due to the same phenomenon as the paper chromatography method, and also the dry state galactose-deficient anti-thyroglobulin monoclonal antibody-immobilized gold colloid develops, and the developed flow reaches the RCA120-coated area No color was observed in the sample using the Tris buffer, and the gold colloid passed through. However, in the sample using the thyroglobulin solution, reddish purple coloration was observed. The procedure is as simple as immersing the absorption pad in the sample in one step, and the time required for the determination is as short as about 4 minutes.Silogroprin can be detected quickly and quickly. INDUSTRIAL APPLICABILITY As described above, the present invention provides a reagent for detecting siloglopurine for the immunochromatography method using RCA120. INDUSTRIAL APPLICABILITY The effects of the present invention that can be used industrially are as follows.

 (1) A detection reagent for use in a detection method capable of accurately, simply, and rapidly detecting a target substance is provided.

 (2) The amount of an anti-galactose-deficient IgG antibody or a glycoprotein having a sugar chain containing terminal galactose and / or —N-acetylgalactosamine in a human body fluid was compared with a conventional assay method. Provided is a detection reagent that can be detected quickly and easily.

 (3) A diagnostic reagent that can accurately, simply, and rapidly diagnose rheumatoid arthritis is provided.

Claims

The scope of the claims 1. A detection reagent for detecting a substance to be detected having a sugar chain containing a galactose and / or ^ or -N-acetylgalactosamine at a terminal, wherein the membrane has a first reactive substance immobilized thereon, A second reactive substance containing immobilized microparticles, wherein one of the reactive substances is ricinosaminisagglutinin I, and the other of the reactive substances specifically binds to the substance to be detected and is galactose. And ^ -N-acetylgalactosamine, a detection reagent which is a reactive substance having no sugar chain at the terminal. 2. The substance to be detected is an anti-galactose-deficient IgG antibody, and the reactive substance having no sugar chain containing galactose and S-N-acetylgalactosamine at the terminal is galactose-deficient IgG. Item 14. The detection reagent according to Item 1. 3. A detection reagent for detecting an anti-galactose-deficient IgG antibody, which contains a membrane and microparticles, and one of the membrane and the microparticles is immobilized with lisinascominisagglutinin I. The other is a detection reagent in which galactose-deficient IgG has been immobilized. 4. The detection reagent according to claim 3, wherein anti-galactose-deficient IgG is immobilized on the membrane, and ricinus minisagglutinin I is immobilized on the microparticles. 5. The substance to be detected is thyroglobulin, and the reactive substance having no sugar containing galactose and 3-N-acetylgalactosamine at its terminal is a galactose-deficient anti-thyroglobulin antibody. The detection reagent as described. 6. A diagnostic agent for rheumatoid arthritis, comprising the detection reagent according to claim 3 or 4. 7. A step of reacting a membrane on which one of galactose-deficient IgG and ricinus cominisagglutinin I is immobilized, a colored microparticle on which the other is immobilized, and a biological sample collected from a subject. When, Binding of anti-galactose-deficient IgG antibody and galactose-deficient IgG contained in the sample, and binding of ricinus-cominisagglutinin I and anti-galactose-deficient IgG antibody Detecting colored fine particles trapped on the membrane through binding to galactose contained therein. A method for detecting rheumatoid arthritis.