JPH0587814A - Method and medicine for rheumatism diagnosis and determination method for agalactosill igg - Google Patents

Abstract

PURPOSE:To increase correctness of diagnosis by measuring the amount of agalactosill IgG (man immunity globulin G) present in man's serum by sandwiching it by man-resistant IgG antibody and rectine. CONSTITUTION:A man-resistant IgG antibody can be bonded chemically and/or physically and directly or indirectly to a porous insoluble carrier. The carrier is desirably to be porous, and diatomaceous earth, titanium dioxide, etc., are given for its material. Next, a rectine is desirably to be ricinus communis agglutinin(RCA) and can be obtained from seed of the ricinus communis by affinity column of beta-D galactose. Then the amount of agalactosill IgG present in man's serum is measured by sandwiching it by man-resistant IgG antibody and rectine. When the amount of agalactosill IgG is larger than the total IgG amount, it is judged that its owner is a rehumatism patient and, when it is smaller than the total IgG amount, it is judged that he is not a rehumatism patient.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to agalactosyl IgG.
It relates to the diagnosis of rheumatism by measuring the amount.

[0002]

BACKGROUND OF THE INVENTION There is an autoimmune antibody called rheumatoid factor in the serum of patients with rheumatoid arthritis, which is known to recognize the Fc portion of human immunoglobulin G (IgG) as an antigen. By the way, recently, a detailed analysis was performed on the sugar chain present in the Fc portion of IgG in the serum of patients with rheumatism, and as a result, the sugar chain was compared with the sugar chain in the Fc portion of IgG in the serum of healthy subjects. It has been reported that the galactose content is significantly reduced. That is, it was revealed that the sugar moiety in the Fc portion of IgG in healthy human serum is composed of a plurality of sugar chains having different structures, and the abundance ratio between species is almost constant among individuals. However, when examining the sugar moiety in the Fc portion of IgG in the serum of rheumatic patients,
It is composed of multiple types of sugar chains with different structures, and the ratio between types is almost the same among individuals as in the case of healthy individuals, but the galactose content is significantly reduced overall. found. More specifically, three types of sugar chains containing 2 molecules of galactose, 1 molecule and 0 molecule of galactose are present in the sugar portion of the Fc portion of IgG in serum of a healthy person at a ratio of about 2: 2: 1. However, it has been reported that in the sugar moiety at the Fc portion of IgG in the serum of rheumatoid patients, the sugar chains of two kinds of galactose molecules are significantly reduced, and the total number of galactose-deficient sugar chains is significantly increased. .. Therefore, based on this fact, there is a structural abnormality in the sugar chain at the Fc portion of IgG in the serum of patients with rheumatoid arthritis, and if it is possible to grasp this structural abnormality, it is due to that of the rheumatoid factor. It can be used as a marker. Then, a measurement for grasping the galactose deficiency in the sugar moiety of the Fc portion of IgG in serum is useful for the diagnosis of rheumatism, but in order to facilitate this measurement, galactose is deficient as a model substance of the measured object. IgG with modified sugar chains, so-called agalactosyl IgG
Is desired to be prepared. When such agalactosyl IgG is prepared, its monoclonal antibody can be prepared, and it can be used to more easily diagnose rheumatism. Rheumatoid arthritis can be diagnosed by directly measuring the rheumatoid factor in serum using the agalactosyl IgG.

Research has been conducted from such a viewpoint,
It was reported that agalactosyl IgG having predetermined physicochemical properties was obtained by treating human IgG with β-galactosidase (JP-A-3-487).
No. 00). However, in the diagnosis according to this proposal, in which a rheumatoid factor is directly measured by a rheumatoid diagnostic agent in which agalactosyl IgG is immobilized on a nitrocellulose membrane, the reaction between the agalactosyl IgG and the rheumatoid factor is not sufficient, and the diagnostic accuracy is high. There is a problem in terms of sex.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to propose a technique capable of accurately diagnosing rheumatism. The object of the present invention is achieved by a rheumatoid diagnostic method characterized by diagnosing by measuring the amount of agalactosyl IgG present in human serum by sandwiching it with an anti-human IgG antibody and a lectin.

Further, it is achieved by a rheumatism diagnostic agent comprising an anti-human IgG antibody and a lectin, which is characterized by sandwiching and measuring the amount of agalactosyl IgG present in human serum. In addition, a galactosyltransferase and UDP-Gal (uracil deoxynucleotide phosphate-galactose) were added to a solution containing human IgG, and the amount of newly transferred galactose was measured to achieve a method for quantifying agalactosyl IgG. To be done.

The anti-human IgG antibody preferably does not contain an Fc portion, the lectin is preferably ricinus cominis agglutinin (RCA), and the above-mentioned anti-human IgG antibody is preferably The lectin immobilized on a carrier is preferably labeled with an enzyme such as horseradish peroxidase. That is,
Since the galactose content of sugar chains existing in the Fc portion of IgG in serum is remarkably reduced in rheumatism patients, quantification of the amount of agalactosyl IgG in serum of rheumatism patients enables accurate diagnosis of rheumatism. Of.

The present invention will be described in detail below. As the anti-human IgG antibody of the present invention, a commercially available product can be used, and it can be directly or indirectly bound to a porous insolubilized carrier chemically and / or physically. Regarding the immobilization method, published in 1976 by Kodansha, Ichiro Chibata et al., "Experimental and Applied Affinity Chromatography" (first edition), 1975, published by Kodansha, Makoto Yamazaki and 2 others "Affinity Chromatography" ( You can refer to the 1st edition). After the binding reaction, a protein that is not involved in the specific reaction to be measured can be loaded for the purpose of eliminating the non-specific reaction of the anti-human IgG antibody. Typical examples thereof include normal mammalian and avian serum proteins, albumin, skim milk, lactic acid fermentation products, collagen, and degradation products thereof.

The carrier to which the anti-human IgG antibody is physically and / or chemically bound is preferably porous, and examples of the material for such carrier include diatomaceous earth, titanium dioxide, barium sulfate, and oxidation. Examples thereof include various synthetic resins such as zinc, lead oxide, microcrystalline cellulose, silica sand, glass, silica gel, crosslinked dextrin, crosslinked polyacrylamide, agarose, crosslinked agarose and polystyrene.
With the porous carrier made of such a material, the primary shape on which the anti-human IgG antibody is immobilized may be any of particles (beads), rods, and plates.

The lectin used in the present invention is usually obtained by extracting from finely ground seeds, precipitating with ammonium sulfate, and then purifying by sugar affinity chromatography. In particular, ricinus cominis agglutinin (RC
A) is obtained from seeds of Hiba by an affinity column of β-D-galactose. The labeling substances used for labeling such lectins include enzymes, enzyme substrates, substances that change the activity of enzymes and enzyme precursors (enzyme inhibitors, prosthetic groups, coenzymes), enzyme precursors, and apozymes. Enzymes, fluorescent substances, etc., peroxidase,
Horseradish peroxidase is particularly preferable. And, the binding of the labeling substance to the lectin can be carried out by known reagents and methods known to those skilled in the art, for example, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, “Enzyme Immunoassay (3rd.
Ed.), Ikusho Shoin, 1987 ”and the Japanese Society for Clinical Pathology,“ Clinical Pathology ”Extra Edition Special Issue No. 53“ Immunoassays for Clinical Testing-Technology and Applications-, Clinical Pathology Publication Society, 198 ”.
For example, the method described in “3 years” can be referred to.

The amount of agalactosyl IgG present in human serum when sandwiched between the above anti-human IgG antibody and lectin is measured as follows. That is, IgG in rheumatism patient serum containing agalactosyl IgG was reacted with an anti-human IgG antibody immobilized on a carrier, B / F separation was performed, and then labeled lectin (RC
React A). After B / F separation again, the signal of the label is detected by an appropriate means.

The signal caused by the label can be detected by an absorbance method (colorimetric method), a fluorescence method or a luminescence method. As a measuring method, a rate measuring method for measuring a change with time of the signal or after a certain period of time is used. Can be measured by an endpoint measurement method that measures the signal of. Then, from the data of the amount of agalactosyl IgG present in the human serum thus measured, it is determined whether or not the patient is a rheumatic patient.
Specifically, it is determined that the patient has rheumatics when the amount of agalactosyl IgG is larger than the total amount of IgG, and the patient is not rheumatic when the amount of agalactosyl IgG is smaller than the total amount of IgG.

[0012]

The present invention will be described in detail below.
The present invention is not limited thereby. [Example 1] Human IgG (35 mg / ml) was added to 0.1 M
Citrate buffer (pH 5.5) 1 ml of sialidase (0.5 mg / ml), followed by sufficient reaction with β-galactosidase (200 mU / ml), and then 1.5 M glycine-HCl, 3 M NaCl (pH
8.9) was added to Protein A-Sepharose CL-4B equilibrated with the buffer. 1.5M Glycine-H
Cl, washed with a buffer of 3M NaCl (pH 8.9), and then washed with 0.1M glycine-HCl (pH 3.
At 0), the agalactosyl IgG sample was collected and dialyzed against phosphate buffer.

Next, human IgG and agalactosyl IgG obtained as described above were mixed with 0:10, 1: 9, 3:
The mixture was mixed at a ratio of 7, 5: 5, 7: 3, 9: 1 and 10: 0 and diluted with a 1% BSA-PBS solution to give a final concentration of the total amount of human IgG and agalactosyl IgG of 50 μg / m 2.
It was set to l. Next, goat-derived anti-human IgG F (ab ')
₂ (Rockland) diluted with PBS to 10 μg / m
100 μl was dispensed into a 96-well immunoplate and physically adsorbed at 4 ° C. overnight. After adsorption, discard the liquid,
After washing with PBS, add 1% BSA-PBS solution to 200
Each μl was added and blocking was performed for 1 hour. After discarding the liquid, it was diluted 100 times. Then, this human IgG solution was diluted 100-fold with PBS and added in 100 μl increments.
The reaction was carried out at 7 ° C for 1 hour. After the reaction is completed, the liquid is discarded and PB is added.
Wash with S and with horseradish peroxidase (HRP)
Labeled RCA HRP (EY Laboratories) and a 1% BSA-PBS solution were added and reacted at room temperature for 1 hour. Also, instead of RCA / HRP, anti-human IgG / HRP
And 1% BSA-PBS solution were added and reacted at room temperature in the same manner. After washing with PBS, 0.02% H ₂ O ₂ and 3 mg /
100 μl of citrate-phosphate buffer (pH 5.0) containing 100 ml of o-phenylenediamine solution was added for color development, and then 50 μl of 9N H ₂ SO ₄ was added for each reaction to stop the reaction. The absorbance at 492 nm was measured, and the results are shown in FIG. According to this, as the amount of agalactosyl IgG decreases, the reaction with lectin increases, the absorbance increases, and a calibration curve can be created.

Next, 12 sera of rheumatic patients and 11 sera of healthy individuals were measured in the same manner as above. That is, 1% BSA-PB was used instead of the human IgG solution.
The same procedure was performed by adding 100 μl of each of rheumatism patient serum and healthy subject serum diluted 20,000 times with S solution. The measurement results are as shown in FIG. 2, and although the total amount of IgG was almost the same, as shown in the calibration curve of FIG. 1, sandwiched between the anti-human IgG antibody and the lectin based on the difference in the amount of agalactosyl IgG. Absorbance of Rheumatoid Patients
The OD is much lower than the absorbance OD of a healthy person, and it is possible to judge from this difference whether or not the patient has rheumatism.

[Example 2] [Binding of anti-IgG polyclonal antibody F (ab ') ₂ to gel] Trisacryl gel GF-2000 (Pierce Chemical Co.) activated with carbodiimide was combined with distilled water and a binding buffer (0. Wash twice with 1M borate solution, pH 8.5). Anti-human IgG polyclonal antibody F (ab ')
GF-20 washing with binding buffer 2ml to ₂ 4 mg
00 gel and shake culture at 4 ° C. overnight. The unreacted active groups of the gel are blocked by incubation with 2 ml of 5% BSA / binding buffer overnight at 4 ° C. The gel with the antibody bound to 0.
1M citrate buffer, 1.4M sodium chloride (p
H4.0) and 0.1M carbonate buffer, 1.4M sodium chloride (pH 11.0) alternately washed, and finally PB
Wash with S. The gel is 5 ml PBS (0.05% Na
And stored in containing the N _3).

[Reaction with GalT] 50 μl of anti-IgG
The gel suspension bound with the antibody and the agalactosyl IgG (20, 10, 5, 1, 0.5 μg) obtained by treating human IgG with β-galactosidase were combined and shaken at 4 ° C. overnight. Cultured. 2 ml cold CKT buffer (20 mM sodium cacodylate, 150 mM
KCl, 0.01% Triton X-100) three times and then the supernatant is aspirated. 100 μl GalT
Reaction solution (5 μl of 0.5 mM [ ³ H] UDP-Gal
Solution, New England Nuclear Co., Ltd.), 1 μl of 1.0 M manganese dioxide solution, 94 μl of CKT buffer and 1.0 U of bovine galactosyltransferase (manufactured by Sigma) are added, and the mixture is incubated at 37 ° C. for 30 minutes. After completion of the reaction, the plate was washed 5 times with 2 ml of a PBS solution containing 0.05% Tween-20, and finally suspended in 200 μl of PBS. Since 100 μl was taken out from the gel suspension and suspended in a liquid scintillator and the radioactivity was measured,
The result is shown in FIG.

According to this, it is understood that the quantification of agalactosyl IgG is possible.

[0018]

[Effect] The rheumatism can be accurately diagnosed.

[Brief description of drawings]

[FIG. 1] Human IgG / Agalactosyl human IgG on the horizontal axis
A graph showing the amount and the absorbance on the vertical axis.

FIG. 2 is a graph showing the absorbance of sera of rheumatism patients and sera of healthy people.

FIG. 3 is a graph showing the quantification of agalactosyl IgG.

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[Procedure amendment]

[Submission date] December 2, 1991

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Claim 6

[Correction method] Change

[Correction content]

[Procedure Amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0005

[Correction method] Change

[Correction content]

Further, it is achieved by a rheumatoid diagnostic agent comprising an anti-human IgG antibody and a lectin, which is characterized by sandwiching and measuring the amount of agalactosyl IgG present in human blood. In addition, galactosyltransferase and UDP-Gal ( uridine-5 ′) were added to a solution containing human IgG.
- diphosphate - galactose) was added, is accomplished by quantitative method agalactosylated IgG, which comprises measuring the amount of newly metastasized galactose.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0013

[Correction method] Change

[Correction content]

Next, human IgG and agalactosyl IgG obtained as described above were mixed with 0:10, 1: 9, 3:
The mixture was mixed at a ratio of 7, 5: 5, 7: 3, 9: 1 and 10: 0 and diluted with a 1% BSA-PBS solution to give a final concentration of the total amount of human IgG and agalactosyl IgG of 50 μg / m 2.
It was set to l. Next, goat-derived anti-human IgG F (ab ')
₂ (Rockland) diluted with PBS to 10 μg / m
100 μl was dispensed into a 96-well immunoplate and physically adsorbed at 4 ° C. overnight. After adsorption, discard the liquid,
After washing with PBS, add 1% BSA-PBS solution to 200
Each μl was added and blocking was performed for 1 hour. After discarding the liquid, dilute the above IgG solution 100 times with PBS and
Each 100 μl was added, and the mixture was reacted at 37 ° C. for 1 hour. After completion of the reaction, the liquid was discarded, washed with PBS, and labeled with horseradish peroxidase (HRP) RCA.HRP (E.
Y. Laboratories) and 1% BSA-PBS solution were added and reacted at room temperature for 1 hour. Also, instead of RCA.HRP, anti-human IgG.HRP and a 1% BSA-PBS solution were added and similarly reacted at room temperature. After washing with PBS, 0.
After adding 100 μl each of citrate-phosphate buffer (pH 5.0) containing 02% H ₂ O ₂ and 3 mg / ml o-phenylenediamine solution to develop color, 9N H ₂ SO ₄ was added.
Was added to each well to stop the reaction, and the absorbance at 492 nm in each well was measured. The results are shown in FIG.
Shown in. According to this, as the amount of agalactosyl IgG decreases, the reaction with lectin increases, the absorbance increases, and a calibration curve can be created.

Claims (6)

[Claims] 1. Agalactosyl I present in human serum
A method for diagnosing rheumatism, which comprises diagnosing by measuring the amount of gG sandwiched between an anti-human IgG antibody and a lectin. 2. The method for diagnosing rheumatism according to claim 1, wherein the anti-human IgG antibody does not contain an Fc portion. 3. The rheumatism diagnostic method according to claim 1, wherein the lectin is ricinus cominis agglutinin (RCA). 4. An anti-human IgG antibody is immobilized on a carrier,
The method for diagnosing rheumatism according to claim 1, wherein the lectin is enzyme-labeled. 5. A rheumatoid diagnostic agent comprising an anti-human IgG antibody and a lectin, which is measured by sandwiching the amount of agalactosyl IgG present in human serum. 6. A method for quantifying agalactosyl IgG, which comprises adding galactosyltransferase and uracil deoxynucleotide phosphate-galactose to a solution containing human IgG, and measuring the amount of newly transferred galactose.