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WO1996024845A1 - Reactif de detection de substances et procede de diagnostic de la polyarthrite rhumatoide - Google Patents

Reactif de detection de substances et procede de diagnostic de la polyarthrite rhumatoide Download PDF

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Publication number
WO1996024845A1
WO1996024845A1 PCT/JP1996/000288 JP9600288W WO9624845A1 WO 1996024845 A1 WO1996024845 A1 WO 1996024845A1 JP 9600288 W JP9600288 W JP 9600288W WO 9624845 A1 WO9624845 A1 WO 9624845A1
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WO
WIPO (PCT)
Prior art keywords
galactose
immobilized
membrane
deficient
substance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1996/000288
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English (en)
Japanese (ja)
Inventor
Atsushi Sakuraoka
Ikue Aonuma
Yoshitsugu Harada
Yuji Yamada
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eisai Co Ltd
Morinaga Milk Industry Co Ltd
Original Assignee
Eisai Co Ltd
Morinaga Milk Industry Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Eisai Co Ltd, Morinaga Milk Industry Co Ltd filed Critical Eisai Co Ltd
Priority to EP96901979A priority Critical patent/EP0756173A4/fr
Publication of WO1996024845A1 publication Critical patent/WO1996024845A1/fr
Priority to NO964279A priority patent/NO964279L/no
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/686—Anti-idiotype
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • 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.
  • an anti-galactose-deficient IgG antibody an antibody against IgG lacking galactose
  • 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.
  • EIA enzyme immunoassay using an enzyme as a label
  • particle agglutination using microparticles particle agglutination using microparticles
  • agglutination inhibition a coloring method using colored fine particles is known.
  • 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.
  • 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.
  • 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.
  • a typical method of the flow through method a method described in Japanese Patent Application Laid-Open No.
  • LH detection kit manufactured by Futatsubon Gene
  • this method is sometimes called the alias filter method, here it is called the flow-through method.
  • an anti-LH monoclonal antibody 1 is applied to a dinitrocellulose membrane to prepare an antibody-immobilized membrane.
  • 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.
  • 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.
  • 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.
  • 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.
  • Reactive Substance 1 Antibody 1
  • Reactive Substance 2 Antibody 2
  • 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).
  • hCG anti-human chorionic gonadotropin
  • hCG anti-human chorionic gonadotropin
  • 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.
  • 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.
  • strong blue coloring can be visually confirmed at the reaction site on the film.
  • the immunochromatography method using colored fine particles has a short operation time, is easy to operate, and is easy to judge.
  • 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).
  • 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.
  • 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 abbreviation of Turbid metric i band unoassay: quantitative method of RF for spectroscopically measuring the degree of aggregation of latex particles.
  • TIA method abbreviation of Turbid metric i band unoassay: quantitative method of RF for spectroscopically measuring the degree of aggregation of latex particles
  • 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.
  • 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.
  • 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.
  • the sugar chains present at the IgG Fc site in the serum of RA patients 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • RCA 120 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.
  • 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.
  • 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.
  • 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.
  • 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”.
  • 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. .
  • 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.
  • the galactose deficient reactive substance is galactose deficient IgG.
  • the present detection reagent can also be carried out by an immunochromatography method.
  • the 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.
  • a water-soluble polymer such as polyethylene glycol, other stabilizers, and a preservative
  • 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.
  • the detection reagent of the present invention 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.
  • 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.
  • 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.
  • 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. .
  • the sample 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.
  • 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.
  • 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.
  • 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 ⁇
  • 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,
  • 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.
  • 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
  • RCA120 binds to galactose or ⁇ -N-acetylgalactosamine contained in the target substance. As a result, fine particles are captured on the membrane.
  • 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.
  • the film after the determination can be dried and stored for a long time without a change in coloring.
  • 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).
  • BZF separation bound form: B und, B formed by binding of an antibody in an antigen-antibody reaction
  • free form Free, F not bound
  • This is a particularly effective method for detection systems that require
  • 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.
  • 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.
  • 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>.
  • RC A 120 as a reactive substance is immobilized on colored microparticles to obtain an RCA 120 immobilized microparticle suspension.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the reaction principle in the use of the anti-galactose-deficient IgG antibody detection reagent is shown below based on FIG.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Diagnostic reagent for rheumatoid arthritis (RA) rheumatoid arthritis
  • 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.
  • 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.
  • 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).
  • 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.
  • the method for detecting rheumatoid arthritis of the present invention comprises:
  • RA rheumatoid arthritis
  • 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.
  • RA rheumatoid arthritis
  • 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.
  • 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.
  • RA rheumatoid arthritis
  • the operation was complicated and took 4 and a half hours.
  • 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 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.
  • 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 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
  • 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).
  • hydrochloric acid pH 3.0
  • 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).
  • 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 ⁇ l 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).
  • Tris buffer 50 mM Tris-HCl, pH 8.0
  • Tris buffer 50 mM Tris-hydrochloric acid, pH 8.0
  • Tris buffer 50 mM Tris-hydrochloric acid, pH 8.0
  • 1% 833, 0.15 M sodium chloride and 0.02% sodium azide 1% 833, 0.15 M sodium chloride and 0.02% sodium azide
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Tris buffer 50 mM Tris-HCl, pH 8.0
  • 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.
  • 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. .
  • 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.
  • a detection reagent for use in a detection method capable of accurately, simply, and rapidly detecting a target substance is provided.
  • a diagnostic reagent that can accurately, simply, and rapidly diagnose rheumatoid arthritis is provided.

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Abstract

On peut diagnostiquer de manière fiable et rapide la polyarthrite rhumatoïde, etc., selon un procédé comprenant les étapes consistant à faire réagir une membrane sur laquelle est immobilisée une IgG déficiente en galactose et une agglutinine I de Ricinnus communis, le second des deux agents précités étant immobilisé sur de fines particules colorées, et un échantillon biologique prélevé sur un sujet, et à détecter ces fines particules colorées capturées sur la membrane par l'intermédiaire de la liaison entre l'anticorps IgG déficient en anti-galactose contenu dans l'échantillon et l'IgG déficiente en galactose, et de la liaison entre l'agglutinine I de Ricinnus communis et le galactose contenu dans l'anticorps IgG déficient en anti-galactose.
PCT/JP1996/000288 1995-02-10 1996-02-09 Reactif de detection de substances et procede de diagnostic de la polyarthrite rhumatoide Ceased WO1996024845A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP96901979A EP0756173A4 (fr) 1995-02-10 1996-02-09 Reactif de detection de substances et procede de diagnostic de la polyarthrite rhumatoide
NO964279A NO964279L (no) 1995-02-10 1996-10-09 Deteksjonsreagenser for sporsubstanser, samt fremgangsmåte ved påvisning av rheumatoid artritt

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JP7/23091 1995-02-10
JP2309195 1995-02-10

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WO1996024845A1 true WO1996024845A1 (fr) 1996-08-15

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113311167A (zh) * 2021-05-12 2021-08-27 广州一步医疗科技有限公司 一种具有偶联凝集素的琼脂糖凝胶的制备方法及试剂盒

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03500925A (ja) * 1987-11-10 1991-02-28 ジー．ディー．サール　アンド　カンパニー タンパク質のグリコシル化のアツセイ
JPH0348700A (ja) * 1989-07-14 1991-03-01 Fujita Gakuen アガラクトシルＩｇＧおよび診断薬
JPH0587814A (ja) * 1991-09-30 1993-04-06 Konica Corp リウマチ診断方法及びリウマチ診断薬並びにアガラクト シルＩｇＧの定量方法
JPH06213887A (ja) * 1991-05-20 1994-08-05 Consiglio Nazi Ricerche チログロブリン含有量決定方法およびこの方法用のキット

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03500925A (ja) * 1987-11-10 1991-02-28 ジー．ディー．サール　アンド　カンパニー タンパク質のグリコシル化のアツセイ
JPH0348700A (ja) * 1989-07-14 1991-03-01 Fujita Gakuen アガラクトシルＩｇＧおよび診断薬
JPH06213887A (ja) * 1991-05-20 1994-08-05 Consiglio Nazi Ricerche チログロブリン含有量決定方法およびこの方法用のキット
JPH0587814A (ja) * 1991-09-30 1993-04-06 Konica Corp リウマチ診断方法及びリウマチ診断薬並びにアガラクト シルＩｇＧの定量方法

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Title
BIOCHEMISTRY, Vol. 29, No. 16, (1990) A. THALL et al., "Distribution of Galalpha1 3Galbeta1 4GluNac Residues on Secreted Mammalian Glycoproteins (Thyroglobulin, Fibrinogen and Immunoglobulin G) As Measured by a Sensitive Solid-Phase Radioimmunoassay", p. 3959-3965. *
J. BIOL. CHEM., Vol. 259, No. 15, (1984) R.G. SPIRO et al., "Occurrence of alpha-D-Galactosyl Residues in the Thyroglobulins from Several Species", p. 9858-9866. *
See also references of EP0756173A4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113311167A (zh) * 2021-05-12 2021-08-27 广州一步医疗科技有限公司 一种具有偶联凝集素的琼脂糖凝胶的制备方法及试剂盒

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