JPH0674954A - Anti-beta2-micro globulin derivative substance antibody and immunity measuring method for beta2-micro globulin derivative material using the same - Google Patents

Abstract

PURPOSE:To provide a method to measure only a beta2-micro globulin derivative material specifically by obtaining an antibody which does not react on ordinary beta2-micro globulin but reacts only on the beta2-micro globulin derivative material. CONSTITUTION:An antibody is obtained so as to react specifically on the whole or a part of the nineteenth amino acid array from the first one on the N terminal side of beta2-micro globulin. A beta2-micro globulin antibody derivative material is measured by using specific reaction between the present antibody and the beta2-micro globulin antibody derivative material, so that an immunity measuring method for the beta2-micro globulin antibody derivative material can be obtained.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a substance derived from β 2 -microglobulin in human body fluid, which is highly sensitive and immunologically independent of the influence of ordinary β 2 -microglobulin. Method and the antibody used therein.

[0002]

2. Description of the Related Art It is known that long-term hemodialysis patients frequently have bone joint symptoms such as carpal tunnel syndrome due to amyloidosis. Recently, this bone-joint disorder is β2-
It was revealed that it is closely related to amyloid deposition using microglobulin as a precursor protein (Gejyo, F. e.
t.al. Kidney Int. 30.pp 385-390, 1986). It has been speculated that β 2 -microglobulin accumulates in the blood of long-term dialysis patients and becomes amyloid. However, it is known clinically that there is no correlation between blood β2-microglobulin concentration and the frequency of dialysis amyloidosis (Gejyo, F.et.al.N.Engl.J.Med. .314.pp585-586,1
986). In addition, as a result of analysis of dialysis amyloid-cis patient serum and amyloid tissue by two-dimensional electrophoresis and immunoblotting, as compared with ordinary β2-microglobulin, the isoelectric point is on the acidic side and the molecular weight is small. Small β
It has been reported that a 2-microglobulin analogue was detected (Ogawa, H. et.al. Clinical Nephrology. 30.p.
p.158-163, 1988). It was clarified that this β 2 -microglobulin analogue is a substance in which the 17th asparagine residue from the N-terminus is deamidated to aspartic acid.
(Odani, H. et al. Biochem. Biophys. Res. Commun. 16
8, pp1223-1229, 1990). Furthermore, as a result of analyzing amyloid kidney stones, the presence of not only normal β2-microglobulin but also a fragment of β2-microglobulin cleaved at a lysine residue was reported (Linke, RP et al. Kidoney.
Int. 36, pp 675-681, 1989). Furthermore, in the serum of patients with rheumatoid arthritis, systemic lupus erythematosus, and small cell lung cancer, cleavage at any position between the 55th serine and the 60th tryptophan from the N terminus of β2-microglobulin normal in serum of patients. The presence of the received β2-microglobulin and the measuring method have been reported. (Mogens Horst Nitssen et al., JP 62-246596).

As described above, in the blood of long-term dialysis patients or patients with immune disorders, lung cancer, etc., normal β2
-In addition to microglobulin, it has been suggested that β2-microglobulin analogues that have undergone some modification such as cleavage exist.

[0004]

Conventionally, as a method for measuring β 2 -microglobulin, human-derived β 2 -microglobulin has been administered to a host animal in an undenatured state, and an antibody collected from this host is used. It depends on the immunoassay used. However, according to this method, since the antibody used was prepared by using intact β2-microglobulin as an immunogen, it naturally follows that it also reacts specifically with normal complete β2-microglobulin. Substance derived from microglobulin, that is β 2 −
It is not possible to specifically measure only the degradation product of microglobulin.

Therefore, an object of the present invention is to provide an antibody which does not react with normal β 2 -microglobulin but only with β 2 -microglobulin-derived substances, whereby β
It is to provide a method for specifically measuring only 2-microglobulin-derived substances.

[0006]

As a result of earnest research, the inventors of the present invention found that all conventional antibodies produced using β 2 -microglobulin as an immunogen are C of β 2 -microglobulin.
It has been found that the one having the terminal region as the epitope and the one having the N-terminal region as the epitope cannot be obtained by the above conventional method. For the first time, an antibody having the N-terminal region of β2-microglobulin as an epitope was provided. Furthermore, an antibody whose epitope is the N-terminal region of β2-microglobulin does not react with ordinary β2-microglobulin, and therefore, using this antibody, only β2-microglobulin-derived substances can be specifically measured. The present invention has been completed by finding that the above can be achieved.

That is, the present invention provides an antibody which specifically reacts with all or a part of the amino acid sequence from the 1st to 19th amino acids on the N-terminal side of β 2 -microglobulin. In addition, the present invention utilizes the specific reaction between the antibody of the present invention and a β2-microglobulin antibody-derived substance,
-An immunoassay method for a β2-microglobulin antibody-derived substance, which comprises measuring a substance derived from a microglobulin antibody.

According to the present invention, the cause of bone and joint damage due to dialysis amyloidosis is clarified and a method for predicting it is found, and therefore a β 2 -microglobulin-derived substance different from normal β 2 -microglobulin can be detected. Further, from the antibody obtained in the present invention, serum, spinal fluid, urine, ascites in many diseases other than dialysis amyloidosis,
It is possible to detect β 2 -microglobulin-derived substances in the pleural effusion and to analyze each pathological condition.

It was confirmed as follows that all the antibodies prepared by using β2-microglobulin as an immunogen have an epitope in the C-terminal region of β2-microglobulin. That is, a peptide corresponding to the amino acid sequence of β 2 -microglobulin shown below was prepared by chemical synthesis. Peptide 1; a peptide corresponding to the first to sixth amino acid sequences from the N-terminus. That is, Ile-Gln-Arg-Th
r-Pro-Lys peptide 2; a peptide corresponding to the 10th to 19th amino acid sequences from the N-terminus. That is, Tyr-Ser-Ar
g-His-Pro-Ala-Glu-Asn-Gly-Lys peptide 3; a peptide corresponding to the 92nd to 99th amino acids from the N-terminal, that is, the 2nd to 9th amino acids from the C-terminal. That is, Lys-Ile-Val-Lys-Trp-Asp-
Arg-Asp peptide 4; a peptide corresponding to the 1st to 10th amino acid sequences from the N-terminus. That is, Ile-Gln-Arg-
Thr-Pro-Lys-Ile-Gln-Val-Tyr Each of the above peptides was radioisotope-labeled with iodine-125 using Bolton-Hunter reagent. Each of these labeled peptides was mixed with a rabbit anti-β 2 -microglobulin antibody, and the anti-rabbit antibody was further reacted with solid-phased magnetic fine particles. After that, the magnetic particles were separated with a magnet and the radioactivity of iodine-125 bound to the magnetic particles was measured to examine the reactivity between the anti-β 2 -microglobulin antibody and the antigen peptide.

The inventors of the present invention have prepared 20 kinds of commercially available β2
-The antibody reactive site of microglobulin was analyzed by this method. As a result, all of the anti-β 2 -microglobulin antibodies react with peptide 3, that is, the C-terminal peptide from the N-terminal to the 92nd to 99th positions, but the other N-terminal side of peptide 1 and peptide 2 and peptide 4 Was not detected.

The above results indicate that the antibody obtained by using ordinary β2-microglobulin as an immunogen shows β2-microglobulin-derived substances produced by the modification of β2-microglobulin, particularly only the N-terminal portion. It suggests that such a substance may not be recognized as an antigen. Therefore, these β 2 -microglobulin-derived substances cannot be detected by the conventional β 2 -microglobulin measurement method.

The fact that the amino acid sequence on the N-terminal side of β2-microglobulin is not recognized by an antibody obtained using ordinary β2-microglobulin as an antigen means that this portion is structurally located in the internal region of the protein. Or the N-terminal sequence of normal β 2 -microglobulin may not be an immunogen. Then, as will be proved in the Examples described below, the antibody of the present invention produced by using the N-terminal region of β 2 -microglobulin as an immunogen shows normal β 2
-No reaction with microglobulin. Therefore, the normal β2
-It is impossible to detect the N-terminal side sequence of β2-microglobulin even with the use of an antibody against microglobulin, and further, clinical significance by detecting the N-terminal sequence cannot be expected.

The antibody of the present invention comprises the amino acid sequence of the 1st to 19th amino acids in the N-terminal region of β2-microglobulin, that is, Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Tyr-Ser-.
It can be obtained by using a peptide consisting of Arg-His-Pro-Ala-Glu-Asn-Gly-Lys or a part thereof as an immunogen. When a part of the amino acid sequence is used as an immunogen, any part can be used as long as it is a peptide containing 5 or more amino acids. 1 in the example below
The antibody of the present invention was prepared using a peptide having the 6th amino acid from the 6th amino acid and a peptide having the 10th amino acid to the 19th amino acid as an immunogen, but the present invention is not limited to this. For example, the 1st to 10th amino acids Other moieties, such as peptides having the same, can also be used as the immunogen. In order to enhance immunogenicity, it is preferable to immunize an animal with a peptide bound to a high molecular substance such as bovine serum albumin. The method for producing an antibody per se is well known in the art, and specifically described in the Examples below.

The antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. A method for producing a monoclonal antibody has already been established and can be easily produced using the above immunogen.

By using the antibody of the present invention, only the decomposed product of β2-microglobulin can be specifically immunoassayed without measuring normal β2-microglobulin. An immunoassay method utilizing an antigen-antibody reaction is well known in this field, and any conventional method can be adopted in the present invention. For example, if classification is performed based on the label, enzyme immunoassay, radioimmunoassay, propensity immunoassay, and method using biotin, and if classified by reaction mode, both sandwich method and competition method can be adopted, These are all well known.

The sample to which the immunoassay method of the present invention can be applied may be any sample for which it is desired to measure β 2 -microglobulin-derived substances, such as long-term dialysis patients or patients with immunological disorders, lung cancer, etc. Blood, serum, cerebrospinal fluid, urine, ascites, pleural effusion and the like.

Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

[0018]

Example 1 Preparation of antiserum against N-terminal peptide The following N-terminal peptide was chemically synthesized. Peptide 1; Ile-Gln-Arg-Thr-Pro-Lys Peptide 2; Tyr-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-Ly
s Next, these peptides were bound to bovine serum albumin in a weight ratio of 1: 2 using glutaraldehyde.
This complex was dissolved in physiological saline at a concentration of 1 mg / ml. This solution was mixed with Freund Complete Adjuvant in an equal amount, and 0.8 mg of the total protein amount per rabbit was administered by intradermal injection. One month later, booster immunization was performed, and two weeks later, blood was collected, and the collection was repeated to obtain antiserum.

Example 2 Quantification of human-derived β 2 -microglobulin-derived substance concentration by radioimmunoassay The concentration of human-derived β 2 -microglobulin-derived substance was measured by a competitive radioimmunoassay using a peptide labeled with iodine 125.

Preparation of labeled peptide Synthetic peptide 2, namely Tyr-Ser-Arg-His-Pro-Ala-Gl
u-Asn-Gly-Lys at a concentration of 1.0 mg / ml in borate buffer (p
H8.5), dissolved in 9.25MBq iodine 125 and BOLTON-
Mix HUNTER reagent (Daiichi Pure Chemicals Co., Ltd.), 4
After incubating at ℃ for 1 hour, Sephadex G
The gel was subjected to simple gel filtration with a -25 (PHARMACIA, SWEDEN) column to remove unreacted iodine before use.

Radioimmunoassay Assay Method As a sample, human serum or urine, or renal cyst fluid collected by surgery from the kidney of a long-term dialysis patient with multiple renal cysts is used. A sample volume of 100 μl was used. After mixing 100 μl of the labeled peptide with the sample, anti-peptide 2 antibody diluted 3000 times with PBS containing 0.3% bovine serum albumin was mixed and incubated for 24 hours. Further, magnetic particles coated with an anti-rabbit immunoglobulin antibody (trade name: Amarex-M, Dynabot) were mixed and allowed to stand for 12 hours, after which BOUND / FREE separation was performed with a magnet. The labeled peptide bound to the magnetic particles was quantified by a gamma counter (ALOKA). As a standard substance, a synthetic curve was prepared using synthetic peptide 2 corresponding to the amino acid sequence from the 10th to the 19th on the N-terminal side (Fig. 1). Also,
This system shows no reaction with synthetic peptide 3 corresponding to the amino acid sequence on the C-terminal side and ordinary β 2 -microglobulin.

The measurement results of the samples are shown in Table 1.

[Table 1] Table 1 Sample β2-microglobulin-derived substance concentration ──────────────────────────── Normal subject urine 1 to 31 ng / ml Renal cyst fluid 250 ng / ml ────────────────────────────

As a result of the measurement, 250 ng / ml β2-microglobulin-derived substance was detected in renal cyst fluid obtained by surgery from the kidney of a long-term dialysis patient who developed polycystic kidney disease. The concentration was 1 to 31 ng / ml, which was a very low concentration compared to renal cyst fluid. Furthermore, this assay system showed no reaction to ordinary β2-microglobulin.

Example 3 Quantification of human-derived β 2 -microglobulin-derived substance concentration by competitive enzyme-immuno-immunoassay It was measured.

Preparation of Assay Kit The assay kit consists of the following components. Biotin-labeled peptide Anti-Peptide 2 antibody Solid phase microtiter plate Reagent A; Reaction buffer solution: 0.005% TWEEN-80 and 0.5% bovine serum albumin-containing phosphate buffer solution (pH 7.5) Reagent B: Wash solution; 0.005% TWEEN-80 Phosphate buffer solution (pH
7.5) Horseradish peroxidase-conjugated avidin-biotin complex (VECTASTAIN) ortho-phenylenediamine solution; 1 tablet of ortho-phenylenediamine (SIGMA) in dilution buffer (0.1M sodium phosphate
Dissolve in 10 ml of -50 mM citric acid-0.05% ferriox (pH 5.0) and mix with 300 μl of 0.6% hydrogen peroxide solution. 1.5 normal sulfuric acid prepared immediately before use

Preparation of labeled peptide Synthetic peptide 2, namely Tyr-Ser-Arg-His-Pro-Ala-Glu-
Asn-Gly-Lys was dissolved in 100 mM sodium carbonate buffer (pH 8.5) at a concentration of 3.5 mg / ml, and 1/10 amount of biotinylation reagent (Su
lfosuccinimidyl 6- (biotinamido) Hexanoate, PIRCE
The product was mixed and incubated at room temperature for 2 hours, and then the reaction was stopped with 2.7 mg of glycine. Purification was performed by reverse phase chromatography using ODS120T (Toso). The column was equilibrated with 0.1% trifluoroacetic acid and 1% acetonitrile, and then the labeled peptide mixture was added. Elution was performed with a linear concentration gradient of 0.1% trifluoroacetic acid and 80% acetonitrile, and the peak was fractionated using the absorbance at 220 nm as an index.

Preparation of Anti-Peptide 2 Antibody Solid Phase Microtiter Plate Anti-Peptide 2 antiserum was ammonium sulfate precipitated, DEAE
-Immunoglobulin G fraction was obtained by ion exchange chromatography using cepharose, and then the anti-BSA antibody component was solid-phase absorbed. It was diluted to 3 μg / ml with a sodium carbonate buffer (pH 8.5), 100 μl was added to a 96-well microtiter plate (NUNC), and incubated overnight at 4 ° C. After washing with reagent B, blocking was performed with reagent A. It was used by washing with reagent B immediately before use.

As the enzyme immunoassay sample, human serum, urine, or renal cyst fluid collected by surgery from the kidney of a long-term dialysis patient with multiple renal cysts was used. The sample was diluted with Reagent A before use. 100 μl of the diluted sample was added to the anti-peptide 2 antibody solid-phase microtiter plate, incubated at room temperature for 10 minutes, and 100 μl of biotin-labeled peptide diluted 2000-fold with reagent A was added and mixed. After reacting at 37 ° C for 30 minutes, it was washed with reagent B. Horseradish peroxidase-conjugated avidin-biotin complex (VECTASTAIN) is diluted 1000 times with reagent B, and 100 μl is added. After incubating at 37 ° C for 30 minutes, it was washed with reagent B. After adding 100 μl of the orthophenylenediamine solution, incubate at room temperature for 30 minutes,
The reaction was stopped with 1.5 N sulfuric acid. The absorbance of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm. The calibration curve was prepared using unlabeled peptide 2 (Fig. 2).

The results of measuring the samples are shown in Table 2.

[Table 2] Table 2 Sample β2-microglobulin-derived substance concentration ──────────────────────────── Normal urine 0 to 2 ng / ml Renal cyst fluid 2000 ng / ml Normal serum 2 ng / ml ───────────────────────────── Measurement results, multiple kidneys 2000ng / ml β2-microglobulin-derived substance was detected in renal cyst fluid collected from the kidney of a long-term dialysis patient who developed cystosis, but the substance is very low in urine and serum of normal subjects. Only detected in concentration. Furthermore, this assay system showed no reaction to ordinary β2-microglobulin.

Example 4 Enzyme immunotherapy
Human-derived β2-microglobulin by Immunoassay
Quantitation of Derived Substances (1) The concentration of human-derived β 2 -microglobulin-derived substances was measured by the sandwich method enzyme immunoassay in which anti-peptide 2 antibody was immobilized and the same antibody was labeled with biotin as a secondary antibody. .

Preparation of Assay Kit The assay kit consists of the following components. Standard substance, peptide 5; peptides 1 to 19 from the N-terminal of the amino acid sequence of β 2 -microglobulin. Ile-Gln-Arg-Thr-Pro-Lys-Ile-Gln-Val-Ty
r-Ser-Arg-His-Pro-Ala-Glu-Asn-Gly-Lys Anti-Peptide 2 Antibody Solid Phase Microtiter Plate Biotin-Labeled Anti-Peptide 2 Antibody Reagent A; Reaction Buffer; 0.005% TWEEN-80 and 0.15% Gelatin Containing phosphate buffer (pH 7.5) Reagent B: Washing solution; 0.005% TWEEN-80 containing phosphate buffer (pH
7.5) Horseradish peroxidase-conjugated avidin-biotin complex (VECTASTAIN) orthophenylenediamine solution; same as in Example 3. 1.5 Normal sulfuric acid

Preparation of anti-peptide 2 antibody solid phase microtiter plate Anti-peptide 2 antiserum was ammonium sulfate precipitated, DEAE
-Immunoglobulin G fraction was obtained by ion exchange chromatography with Sepharose, and then the anti-BSA antibody component was solid-phase absorbed. It was diluted to 3 μg / ml with a sodium carbonate buffer (pH 8.5), added to a 96-well microtiter plate (NUNC) in an amount of 100 μl, and incubated overnight at 4 ° C. After washing with reagent B, blocking was performed with reagent A. It was used by washing with reagent B immediately before use.

Preparation of biotin-labeled anti-peptide 2 antibody Anti-peptide 2 antiserum was ammonium sulfate precipitated and DEAE
-Immunoglobulin G fraction was obtained by ion exchange chromatography using cepharose, and then the anti-BSA antibody component was solid-phase absorbed. After dialysis against carbonate buffer (pH 8.5),
Sulfosuccinimidyl 6- (biotinamido) Hexanoate was added at a 1/5 volume and incubated at room temperature for 2 hours. 17
After stopping the reaction with 10 mg of double glycine, 20 mM Tris-hydrochloric acid, 150 mM sodium chloride-0.1% sodium azide (pH
7.5) was dialyzed.

As an enzyme immunoassay sample, human serum or urine, or renal cyst fluid collected by surgery from the kidney of a long-term dialysis patient with multiple renal cysts was used. The sample was diluted with Reagent A before use. Reagent A on anti-peptide 2 antibody solid-phase microtiter plate
Add 100 μl of sample or standard solution diluted with
Incubate overnight at 4 ° C and wash with Reagent B. reagent
Biotin labeled anti-peptide 2 antibody diluted 1000 times with A 1
Add 00 μl and mix. After reaction at 37 ° C for 30 minutes, reagent B
Washed with. Horseradish peroxidase-conjugated avidin-biotin complex (VECTASTAIN) was diluted 1000-fold with reagent B and 100 μl was added. After incubating at 37 ° C for 30 minutes, wash with reagent B. After adding 100 μl of the orthophenylenediamine solution, incubate at room temperature for 10 minutes,
Stop the reaction with 1.5N sulfuric acid. The absorbance of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm.

The calibration curve shows that peptide 5; β is used as a standard substance.
It was prepared using the peptides from the N-terminal to the 19th amino acid of 2-microglobulin (Fig. 2). In addition, this assay system uses peptide 2 (β 2 -microglobulin from the 10th to 19th position from the N-terminal of the amino acid sequence.
No.) but no reaction to normal β 2 -microglobulin was detected.

Table 3 shows the results of measurement of human serum, urine, renal cyst fluid collected from the kidney of a long-term dialysis patient with polycystic renal cyst disease by operation, and serum of dialysis amyloidosis patient as samples.

[Table 3] Table 3 Sample β2-microglobulin-derived substance concentration ──────────────────────────── Normal subject urine 0 to 1 ng / ml Renal cyst fluid 10000 ng / ml Normal serum 0 to 1 ng / ml Dialysis amyloidosis Patient serum 130 ng / ml ───────────────────────── ───── As a result of the measurement, the renal cyst fluid collected by surgery from the kidney of a long-term dialysis patient who developed polycystic kidney disease was 10,000 ng / ml.
The substance derived from β2-microglobulin was detected. Also,
130 ng / ml β 2 − in serum of dialysis amyloidosis patients
A substance derived from microglobulin was detected. However, the substance was detected only at a very low concentration in urine and serum of normal persons.

Example 5 San-Germany Method Enzyme Immuno
Human-derived β2-microglobulin by Immunoassay
Quantitation of Derived Substances (Part 2) The concentration of human-derived β2-microglobulin-derived substances was measured by the sandwich method enzyme immunoassay using an anti-peptide 1 antibody as a solid phase and a biotin-labeled anti-peptide 2 antibody as a secondary antibody. .

Preparation of Assay Kit The assay kit consists of the following components. Standard substance, peptide 5; peptides 1 to 19 from the N-terminal of the amino acid sequence of β 2 -microglobulin. Anti-Peptide 1 Antibody Solid Phase Microtiter Plate Biotin-Labeled Anti-Peptide 2 Antibody Reagent A; Reaction Buffer; 0.005% TWEEN-80 and 0.15% Gelatin-Containing Phosphate Buffer (pH 7.5) Reagent B: Washing Solution; 0.005% TWEEN- 80-containing phosphate buffer (pH
7.5) Horseradish peroxidase-conjugated avidin-biotin complex (VECTASTAIN) orthophenylenediamine solution; same as in Example 3. 1.5
Normal sulfuric acid

Preparation of anti-peptide 1 antibody solid phase microtiter plate Anti-peptide 1 antiserum was ammonium sulfate precipitated, DEAE
-Immunoglobulin G fraction was obtained by ion exchange chromatography using cepharose, and then the anti-BSA antibody component was solid-phase absorbed. It was diluted to 5 μg / ml with sodium carbonate buffer (pH 8.5), 100 μl was added to 96-well microtiter plate (NUNC), and incubated overnight at 4 ° C. After washing with reagent B, blocking was performed with reagent A. It was used by washing with reagent B immediately before use.

Preparation of biotin-labeled anti-peptide 2 antibody Preparation was carried out in the same manner as in Example 4.

As an enzyme immunoassay sample, human serum or urine, or renal cyst fluid collected by surgery from the kidney of a long-term dialysis patient with multiple renal cysts was used. The sample was diluted with Reagent A before use. Reagent A on anti-peptide 1 antibody solid phase microtiter plate
Add 100 μl of sample or standard solution diluted with
After incubating overnight at 4 ° C., it was washed with reagent B. reagent
10-fold diluted biotin-labeled anti-peptide 2 antibody with A
0ul was added and mixed. After reacting at 37 ° C. for 30 minutes, it was washed with reagent B. Horseradish peroxidase-conjugated avidin-biotin complex (VECTASTAIN) was diluted 1000 times with reagent B,
Add 100 μl. After incubating at 37 ° C. for 30 minutes, it was washed with reagent B. After adding 100 μl of the orthophenylenediamine solution, incubate at room temperature for 30 minutes,
The reaction was stopped with 1.5 N sulfuric acid. The absorbance of each well was measured at a main wavelength of 492 nm and a sub wavelength of 630 nm.

The calibration curve shows that the standard substance is peptide 5; β
It was prepared by using the peptides from the N-terminal to the 19th amino acid of 2-microglobulin. (Fig. 2) In addition, in this assay system, no reaction to ordinary β2-microglobulin was detected.

Table 4 shows the results obtained by measuring human serum or urine, renal cyst fluid collected from the kidney of a long-term dialysis patient with multiple renal cysts by surgery as a sample.

[Table 4] Table 4 Sample β2-microglobulin-derived substance concentration ──────────────────────────── Normal subject urine 0 to 1 ng / ml Renal cyst fluid 5200 ng / ml Normal serum 7 ng / ml Dialysis Amyloidosis Patient serum 20 ng / ml ────────────────────────── As a result of the measurement, 5200 ng / ml β2-microglobulin-derived substance was detected in renal cyst fluid collected from the kidney of a long-term dialysis patient who developed polycystic kidney disease by surgery. In the serum of patients with dialysis amyloidosis, β of 20 μg / ml was used.
A substance derived from 2-microglobulin was detected. But,
The substance was hardly detected in the urine of normal persons. Also, the concentration of β2-microglobulin-derived substance detected in the serum of normal humans was as low as 7 ng / ml.

[0044]

INDUSTRIAL APPLICABILITY The present invention enables rapid and quantitative measurement of β2-microglobulin-derived substances contained in human body fluids and the like without being affected by ordinary β2-microglobulin. In particular, by measuring the body fluid components of long-term dialysis patients, as a marker of the onset of bone joint symptoms such as carpal tunnel syndrome due to amyloidosis,
It becomes possible to carry out more accurate measurement compared to the conventional measurement of β2-microglobulin for grasping the pathological condition of other diseases

[Brief description of drawings]

FIG. 1 is a diagram showing a calibration curve of a radioimmunoassay method by a competitive method using an anti-peptide 2 antibody.

FIG. 2 is a diagram showing a calibration curve of an enzyme immunoassay method by a competitive method using an anti-peptide 2 antibody.

FIG. 3 is also a diagram showing a calibration curve of an enzyme immunoassay method by a sandwich method using an anti-peptide 2 antibody as a solid phase antibody and the same anti-peptide 2 antibody as a labeled antibody.

FIG. 4 is a diagram showing a calibration curve of an enzyme immunoassay method by a sandwich method using an anti-peptide 1 antibody as a solid phase antibody and an anti-peptide 2 antibody as a labeled antibody.

Claims (4)

[Claims] 1. An antibody that specifically reacts with the whole or a part of the amino acid sequence from the 1st to 19th amino acids on the N-terminal side of β 2 -microglobulin. 2. The antibody according to claim 1, which specifically reacts with all or part of the amino acid sequence from the 1st to 10th amino acids on the N-terminal side of β 2 -microglobulin. 3. N-terminal side 10 of β2-microglobulin
The antibody according to claim 1, which specifically reacts with all or part of the amino acid sequence from the 19th position to the 19th position. 4. A β2 comprising measuring the β2-microglobulin antibody-derived substance by utilizing a specific reaction between the antibody according to any one of claims 1 to 3 and the β2-microglobulin-derived substance. -An immunoassay method for a substance derived from a microglobulin antibody.