JPH0753757B2 - Monoclonal antibody and method of using the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a monoclonal antibody having specificity for histidine-rich-glycoprotein (hereinafter abbreviated as HRG) and a method for using the same.

[Conventional technology]

HRG is a single-chain glycoprotein with a molecular weight of 67,000.
In 2 years N. Heimburger et al. [Hop-
Zaire's Zuitoshiyurufuyufuru Fuijiorogitsushie Hemi (Hoppe-Seyler's Z.Physiol.Chem.)
Vol. 353, p. 1133 (1972)], which was first purified from human serum and named HRG because of its abnormally high histidine content.

HRG has been studied from various angles in recent years, including high affinity for heparin, interaction with plasminogen, fibrinogen, thrombospondin, heparin, etc.,
Many properties such as various divalent metal ions, heme, and rose bengal binding ability have been reported, and it has come to be considered as one of multifunctional proteins.

It has been known that changes in blood level of HRG during various diseases are decreased in sepsis, hepatic dysfunction, severe cirrhosis, and increased in myocardial infarction and rheumatic heart disease. Recently, it has been reported that thrombosis tends to occur frequently in families with HRG excess, and it has been known that HRG plays an important role as a regulator of coagulation / fibrinolysis system.

Therefore, measuring HRG in blood has important measurement significance in clinical diagnosis. An immunodiffusion method using an antiserum against HRG has been known as a conventionally known method for measuring HRG.

[Problems to be Solved by the Invention]

However, since the conventional method uses animal antiserum, it is extremely difficult to stably obtain an antiserum having a certain activity, and there is a drawback that immunodiffusion takes a long time.

An object of the present invention is to provide an anti-HRG monoclonal antibody which does not have the above-mentioned drawbacks and a method for using the same.

[Means for Solving the Problems]

Briefly describing the present invention, the first invention of the present invention relates to an anti-HRG monoclonal antibody, which does not cross-react with antithrombin III, plasminogen, fibrinogen, and binds plasminogen, fibrinogen, heparin to HRG. It is characterized in that it is a monoclonal antibody whose reactivity with HRG is not inhibited. The second invention is the measurement of HRG in a biological sample, which comprises the anti-H of the first invention.
The present invention relates to a method for measuring HRG using an RG monoclonal antibody.

As a result of intensive studies to overcome the above-mentioned problems, the present inventors have succeeded in obtaining a monoclonal antibody having specificity for HRG, and by using this monoclonal antibody, specific HRG can be obtained. Moreover, they have found that it is possible to measure easily and have completed the present invention.

The monoclonal antibody of the present invention is produced by a so-called cell fusion method. That is, it is produced by forming a fused hybridoma between an antibody-producing cell and a myeloma cell, cloning the hybridoma, and selecting a clone that produces an antibody showing specificity for HRG. As the antibody-producing cells, for example, B lymphocytes derived from splenocytes or lymph node cells from an animal immunized with HRG can be used. Examples of animals to be immunized include mice, rats, rabbits and the like.

Human blood-derived HRG can be used as the antigen, and for example, human blood plasma is purified by heparin affinity chromatography and ion exchange chromatography, and human HRG thus obtained is mixed with Freund's adjuvant to obtain an animal. Immunize with. Immunization is carried out by administering subcutaneously, intramuscularly or intraperitoneally to the animal. About 2 to 5 days after the final immunization, antibody-producing cells are fractionated from the immunized animal.

As the myeloma cells, those derived from mouse, rat, human and the like are used. Cell fusion can be performed, for example, by G. Khle.
r) et al., Nature, 256, 495 (197).
It is carried out by the method described in 5) or a method equivalent thereto. At this time, 30-50% polyethylene glycol (molecular weight 10
00 to 4000) and reacting at a temperature of 30 to 40 ° C. for about 1 to 3 minutes.

The hybridoma obtained by cell fusion is subjected to screening. That is, screening is performed by the enzyme antibody method or the like. The obtained antibody-producing hybridoma is subjected to cloning. That is, the hybridoma is cloned by, for example, the limiting dilution method to obtain a clone. The obtained clones are then subjected to screening for clones producing the desired monoclonal antibody, for example, by the enzyme antibody method. The selected clone is, for example, B to which pristane (2,6,10,14-tetramethylpentadecane) was previously administered.
ALB / c mouse is transplanted into the abdominal cavity, and 10 to 14 days later, ascites fluid containing a high concentration of the monoclonal antibody is collected. The recovery of the monoclonal antibody from the ascites can be easily achieved by applying the conventionally known ammonium sulfate fractionation method, ion exchange chromatography, gel chromatography, affinity chromatography, etc. as a purification method for Ig.

The anti-HRG monoclonal antibody thus obtained is extremely suitable for specifically and conveniently measuring HRG in a sample derived from a living body, such as serum, blood plasma or urine. For this determination, the monoclonal antibody itself or a fragment from it with corresponding immunological properties, such as F
(ab) ₂ fragments can be used.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Monoclonal Antibody (1) Purification of Antigen Citrated fresh blood plasma (1) was added to polybrene (50).
mg), benzamidine (final concentration 1 mM), soybean tribucin inhibitor (50 mg), PMSF (phenylmethanesulfonyl fluoride, final concentration 0.1 mM) and aprotinin (360
00 Kallikrein Inhibition Unit), then 1M BaCl ₂ 80m
Add l gradually. Allow to stand for 1 hour after the addition, 20 at 7800 × g
Centrifuge for minutes to obtain a supernatant fraction.Add 0.1 M EDTA solution to this to 0.1 mM.
And a saturated fraction of ammonium sulfate of 35% to 80% is obtained by a conventional method. This was redissolved in distilled water, and 0.04 containing 0.4M NaCl.
Dialyze against 30 l of 2M phosphate buffer (pH 6.3) for one day. Then, the dialysis solution was heparin-equilibrated with the above buffer.
Add to an agarose column (6.8 cm x 11 cm) and elute with 0.02 M phosphate buffer (pH 6.3) containing 1 M NaCl.
Elute the adsorbed protein by ~ 3 M NaCl gradient method. A bimodal peak is obtained, and the peaks eluting ahead are collected and dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 10 mM sodium citrate and 20 mM NaCl. This was added to a DEAE-Sephadex A-50 column (2.5 cm x 20 cm) equilibrated with the buffer described above, and 2
Elute the adsorbed protein by the gradient method of 0 to 320 mM NaCl. The peaks eluting earlier were collected and purified HRG was obtained and used as an antigen.

(2) Immunization of mice HRG containing 10 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl
And 1: 1 (v / v) with Freund's complete adjuvant
The above mixture was mixed well and the mice were immunized intraperitoneally so that HRG was 50 μg per mouse. 21 days after the first immunization HRG
50 μg was mixed with Freund's incomplete adjuvant and intraperitoneally administered, and 14 days later, 20 μg of HRG was administered via the tail vein.

(3) Cell fusion and cloning Three days after the final immunization, the mouse spleen was taken out, and the splenocytes and mouse myeloma P3U1 were mixed at a ratio of 10: 1, and cell fusion was performed according to the method of Keller et al. I went. Next, implant in a 96-well microplate and HA
T (hypoxanthine 1 × 10 ^-4 M, aminobuterin 4 × 10
^-7 M, thymidine 1.6 × 10 ⁻⁵ M) in MDEM-10% FCS medium (HAT medium) for 10 to 17 days, and then HT (hypoxanthine 1 × 10 ⁻⁴ M, thymidine 1.6 × 10 ⁻⁵ M). M) containing DMEM-1
Transferred to 0% FCS medium (HT medium) and further flask (25 ml)
After the culture became possible, the culture was continued in DMEM-10% FCS medium. The antibody titer in the culture supernatant of the well in which the proliferation was observed was measured by the enzyme antibody method, and the desired hybridoma was cloned from the appropriate well by the limiting dilution method. That is, about 2.5 per well in the microplate.
X10 ⁴ mouse thymocytes were inoculated, and then the hybridoma was diluted to 5, 1, 0.5 / 0.1 ml with DMEM medium, and 0.1 ml / well was inoculated into the above microplate and cultured. Colonies that were visible to the naked eye were formed 10 to 14 days after the start of culturing to obtain a clone strain.

(4) Screening method The culture supernatant of the well in which the hybridoma and the clone were propagated was collected and the enzyme-linked immunosorbent was used.
Atsushi (Enzyme Linked Immunosorbent Assay) (ELI
The antibody production ability against HRG was examined by the SA method. HRG was dispensed to a microtiter plate at 0.5 μg / 50 μl / well and allowed to stand at 4 ° C. for 18 hours to adsorb HRG to a solid phase. 10 mM phosphate buffered saline (PBS) (pH7.4) 20
After washing each well 3 times with 0 μl, 100 μl / well of PBS containing 1% bovine serum albumin (BSA) was added, and the well was incubated at 37 ° C. for 1 hour.
After allowing to stand for a while, the unadsorbed portion of each well was blocked. Then, 50 μl / well of a culture solution as a sample was added and reacted at 37 ° C. for 1 hour. After washing 3 times with PBS, 50 times diluted 1000 times peroxidase-labeled anti-mouse IgG (manufactured by Kapel)
μl / well was added and reacted at 37 ° C. for 1 hour. After washing with PBS, 0.1M citrate buffer (PH4) containing 0.001% hydrogen peroxide, 0.55 mg / ml ABTS [2,2'-azino-di (3-ethylbenzothiazoline-sulfonate)] (Boehringer Mannheim). .0) was added and the absorbance at a wavelength of 405 nm was measured. In the sample, strong coloration was observed when the antibody against HRG was present.

As a result, clone strains HR-1 and HR-2 with high antibody-producing ability are obtained.
6 was obtained.

The clone strains were designated as Hybridoma HR-1 and were respectively designated as Microindustrial Research Institute No. 10171 (FERM P-10171), Hybridoma HR-26.
It has been deposited with the Institute for Microbial Technology, National Institute of Advanced Industrial Science and Technology, as Microindustry Research Institute No. 10172 (FERM P-10172).

(5) Preparation of monoclonal antibody Clone strains 1 to 9 which were cultured and propagated after intraperitoneal administration of 0.5 ml of pristane (manufactured by Aldrich Co., Ltd.) to BALB / c mice aged 7 weeks or older and 1 week or longer × 10 ⁸ cells / mouse were inoculated intraperitoneally. After 10 to 14 days, the mice were killed and ascites was collected. Centrifuge this at 3,000 rpm for 10 minutes, and
Ascitic fluid containing monoclonal antibody (ml / mouse) was obtained.

(6) Purification of Monoclonal Antibody The ascites obtained in (5) above was filtered with absorbent cotton to remove fat, and diluted 2-fold with 50 mM phosphate buffer (pH 7.3), and then saturated with an equal volume of 100%. Ammonium sulfate was added, and the precipitated fraction was collected. This fraction was dissolved in a small amount of the above phosphate buffer and dialyzed against the buffer. Apply this sample to a DEAE-cellulose column and
HRG monoclonal antibodies HR-1 and HR-26 were obtained.

(7) Physicochemical properties of monoclonal antibody Ig subclass HR-1: IgG ₁ HR-26: IgG ₁ Octaronie gel diffusion test using a specific class of purified monoclonal antibody using class-specific anti-mouse Ig antibody Decided.

Molecular weight HR-26: 170,000 ± 5,000 HR-1: 165,000 ± 5,000 The molecular weight of the monoclonal antibody was estimated by SDS-polyacrylamide gel electrophoresis (12.5%). The molecular weight marker is SDS-PAGE molecular weight standard from Hi-Bio-Hi
gh was used (myosin 200 kDa, β-galactosidase
116 kDa, Phosphorivase b 97 kDa, Serum albumin 6
6kDa, ovalbumin 43kDa).

Reactivity with antigen HR-1: Reacts with CNBr 50kDa fragment HR-26: Does not react with the above fragment HRG is decomposed by CNBr [T. Koide et al., Journal of Biochemistry (J. Biochem. ) Volume 98,
Pp. 1191 to 1200 (1985)], dissolved in 50 mM Tris-HCl buffer pH 8.6, and subjected to SDS-polyacrylamide electrophoresis, and then subjected to immunoblot method [WN Brunet (WNBurnette), Analytical Biochemistry (Anal). Biochem.) 112, 195-203 (198
1)] The reactivity with the monoclonal antibody was examined.

As a result, the N-terminal side of HRG obtained by decomposing HRG with CNBr 50
HR-1 and HR-26 did not react with the kDa peptide.

Each CNBr fragment was identified by the method of T. Koide et al. (Supra).

Cross-reactivity HR-1: Antithrombin III, plasminogen, no fibrinogen cross-reactivity HR-26: Antithrombin III, plasminogen, fibrinogen no cross-reactivity antithrombin III, plasminogen, fibrinogen Were dissolved in PBS to be 10 μg / ml each, and these were each coated on a microtiter plate,
Cross-reactivity with the above substances was examined in the same manner as in the screening method for hybridomas.

Effects of coexisting substances HR-1: HR-26, plasminogen, fibrinogen,
Reactivity with HRG is not affected by the presence of heparin HR-26: HR-1, plasminogen, fibrinogen,
Reactivity with HRG is not affected by the presence of heparin. Add 50 μl / well of HRG (dissolved in PBS to 5 μg / ml) to a microtiter plate, coat at 37 ° C. for 3 hours, wash with PBS, and then 200 μl / well of PBS containing% bovine serum albumin is added and blotted at 37 ° C. for 1 hour and washed with PBS. Add HR-26 (10 μg / m
l), HR-1 (10 μg / ml), plasminogen (10 μg / m
l), fibrinogen (10 μg / ml), heparin (1 IU / m)
l) at 50 μl / well and reacted at 37 ° C for 1 hour, followed by the Nakane method [PK Nakan (PK Nakan)
e), Journal of Histochemistry and Sight Chemistry (J.Histochem.Cytochem) No. 22
Vol. 1084 (1974)]. Peroxidase (POD) -labeled HR-1 or HR-26 was added at 50 μl / well each, reacted at 37 ° C. for 1 hour, washed with PBS, and then the ABT
S is developed as a chromogenic substrate, the absorbance at 405 nm is measured,
The effect of the coexisting substance was examined in comparison with the case where the coexisting substance was not added.

The above results are summarized in Tables 1 and 2. Table 1 shows HR-
1. Ig subclass of HR-26, reactivity with antigen, cross-reactivity, and Table 2 summarizes the effect of coexisting substances. HR-1 and HR-26 are different epitopes on HRG. It was demonstrated that the reactivity of plasminogen, fibrinogen, and heparin was not affected by binding and coexistence with HRG. Especially HR-26 is CNB
It was shown that the reactivity was lost or significantly reduced by r fragmentation.

Example 2 San-Germany EI using a monoclonal antibody
Measurement of HRG by Method A The HRG measurement reagents were prepared using the monoclonal antibodies HR-1 and HR-26 obtained in Example 1.

(1) Preparation of Peroxidase (POD) -Labeled Monoclonal Antibody Monoclonal antibody HR-1 was PO by the Nakane method (supra).
D labeled.

(2) Sangerichi EIA assay system 100 μl of monoclonal antibody HR-26 (10 μg / ml) dissolved in PBS was added to each well of a 96-well microtiter plate.
After adding 1 liter each and incubating overnight at 4 ° C., discarding the solution, 200 μl each of PBS solution containing 1% BSA was added to each well, and blocking was carried out at 37 ° C. for 1 hour. After thorough washing with PBS, add 200 μl of sample and continue 1
100 μl of POD-labeled HR-1 antibody solution diluted with PBS containing% BSA
After reacting at 37 ℃ for 30 minutes, wash with PBS 3 times, and add 0.1M citric acid-sodium hydroxide buffer (pH 4.0) containing 0.001% hydrogen peroxide and 0.55mg / ml ABTS. The absorbance at a wavelength of 405 nm was measured.

(3) Measurement of Specimens Plasma samples obtained from 18 healthy individuals, 18 myocardial infarctions, and 12 cerebral infarctions were diluted 1000-fold with 1% BSA-containing PBS, and measured by the EIA method. At this time, the HRG concentration of each sample was determined from the calibration curve obtained using purified HRG as a standard product. First result
Shown in the figure. That is, FIG. 1 is a diagram showing the relationship between the HRG concentration (μg / ml, vertical axis) and each plasma sample (horizontal axis), which is the HRG measurement result of plasma by the San-Germany EIA method using the monoclonal antibody of the present invention. Is.

In the healthy group, the mean ± SD was 100.6 ± 19.15 μg / ml. On the other hand, in myocardial infarction group and cerebral infarction group, respectively 115.2 ± 51.3 μg / m
l, 117.7 ± 61.6 μg / ml, showing a broader distribution than in healthy subjects, and there were cases in which abnormal values were observed when the mean ± 2SD of the healthy group was used as the cut off value.

Example 3 Measurement of HRG by Latex Aggregation Method Using Monoclonal Antibody The HRG measurement reagents were prepared using the monoclonal antibodies HR-1 and HR-26 obtained in Example 1.

(1) Sensitization of Latex 0.05M glycine-NaOH buffer containing 0.1M NaCl (GB,
pH8.5), latex (manufactured by Sekisui Chemical Co., Ltd.,
Particle size 0.464 μm] 1 volume of suspension dispersed so that the solid content is 4.0% (w / v) and HR-1 or HR-26 (30 mg / ml each)
1 volume of the solution was mixed with each other and incubated at 37 ° C. for 3 hours. GB6 containing BSA, sucrose, choline chloride, NaN ₃
The final concentration of lattex solids 0.5%, BSA1
%, Sucrose 1%, choline chloride 0.1M, NaN ₃ 0.1%. HR-1 and HR-26 were separately sensitized and mixed at a ratio of 1: 1 before use, and this was used as a latex reagent.

(2) Method for measuring sample 20 μl of a sample obtained by diluting a blood sample 1000 times with PBS containing 1% BSA and 20 μl of a latex reagent were mixed on a latex agglutination determination plate, and after 3 minutes, the presence or absence of aggregation was determined.
At this time, the detection sensitivity of this system using purified HRG was 150 ng / ml. Healthy people using this measurement method 14
Examples, cerebral infarction 13 cases, myocardial infarction 10 cases, as a result of measurement using blood sample, as shown in Table 3, cerebral infarction,
It was shown that the blood HRG level was high in patients with myocardial infarction.

[Effects of the Invention] As described in detail above, the present invention provides a monoclonal antibody against HRG. By using the monoclonal antibody of the present invention, HRG is simple and highly specific.
It is very useful for studying the physiological effects of HRG and for diagnosing blood coagulation / fibrinolytic system diseases.

[Brief description of drawings]

FIG. 1 is a diagram showing the results of measurement of the HRG concentration in blood plasma by the San-Germany EIA method using the monoclonal antibody of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 21/08 9161-4B (C12P 21/08 C12R 1:91) (72) Inventor Ikuyuki Kato Shin, 3-4-1 Seta, Otsu City, Shiga Prefecture, Central Research Laboratory, Mina Shuzo Co., Ltd. (56) References Biochemistry, 25 [8] (1986) p. 2220-2225 Eur. J. Biochem. , 119 [3] (1981) p. 641-646 The Journal of Biological Chemistry, 255 [21] (1980) P. 10214-10222

Claims (4)

[Claims] 1. An antihistidine-rich-glycoprotein antibody by binding plasminogen, fibrinogen, heparin to histidine-rich glycoprotein without cross-reacting with antithrombin III, plasminogen, fibrinogen. Anti-histidine-rich-glycoprotein monoclonal antibody whose reactivity is not inhibited. 2. The monoclonal antibody according to claim 1, which has the following properties. (A) Molecular weight: 170,000 ± 5,000 (b) Ig subclass; IgG ₁ (c) Reactivity with antigen: 50 kDa of N-terminal side of histidine-rich-glycoprotein obtained by degrading histidine-rich-glycoprotein by CNBr Does not react with peptides 3. The monoclonal antibody according to claim 1, which has the following properties. (A) Molecular weight: 165,000 ± 5,000 (b) Ig subclass; IgG ₁ (c) Reactivity with antigen: 50 kDa of N-terminal side of histidine-rich-glycoprotein obtained by decomposing histidine-rich-glycoprotein by CNBr React with peptides 4. A method for measuring histidine-rich-glycoprotein, which comprises using the anti-histidine-rich-glycoprotein monoclonal antibody according to claim 1 in measuring histidine-rich-glycoprotein in a biological sample. Method.