JPH0249595A - Monoclonal antibody and use thereof - Google Patents

Abstract

NEW MATERIAL:An antihistidine-rich glycoprotein monoclonal antibody with 170000+ or -5000 molecular weight and IgG1 Ig-subclass capable of recognizing the stereostructure of histidine-rich glycoproteins, resistant to cross reactions with antithrombin III, plasminogen, fibrinogen and free from inhibition of the reactivity due to linkage to plasminogen, fibrinogen or histidine-rich glycoproteins of heparin. USE:A reagent for measurement of histidine-rich glycoproteins. PREPARATION:For example, a human hematogenous histidine-rich glycoprotein is administrated to a mouse, etc., and immunized splenic cells are collected to be fused with myeloma cells. Antibodyproducing clones are selected therefrom and subjected to cloning by the limiting dilution method, etc., for monocloning. The resultant monoclonal fused cells are cultured to obtain a monoclonal antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] 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.
s Z, Physiol, Chem, ) No. 553
Vol., p. 1153 (1972)], it was first purified from human serum and named HRG because of its unusually high histidine content.

HRG has been studied from various angles in recent years, including its high affinity for heparin, interactions with plasminogen, fibrinogen, trompospondin, heparin, etc.
Numerous properties have been reported, including the ability to bind various divalent metal ions, heme, and rose bengal, and it has come to be considered as a multifunctional protein.

It has been known that changes in the blood level of HRG during various diseases decrease in cases of sepsis, liver dysfunction, severe liver cirrhosis, etc., and increase in cases of myocardial infarction, rheumatic heart disease, etc. Recently, it has been reported that families with excess HRG tend to have frequent thrombosis, and it has become known that HR() plays an important role as a regulating factor of the coagulation and fibrinolytic systems.

Therefore, measuring HRG in collapsing fluid has important significance in clinical diagnosis. A conventionally known method for measuring HRG is the immunodiffusion method using antiserum against HRG.

[Problem to be solved by the invention]

However, since the conventional method uses animal antiserum, it is extremely difficult to stably obtain an antiserum with a certain level of activity, and it also has the drawback that it takes a long time for immune diffusion.

An object of the present invention is to provide an anti-HRG monoclonal antibody and a method for its use that are free from the above-mentioned drawbacks.

[Means to solve the problem]

To summarize the present invention, the first invention relates to an anti-ERG monoclonal antibody, and the second invention relates to an anti-ERG monoclonal antibody for measuring HR() in a biological sample. Regarding measurement methods.

As a result of intensive research to overcome the above-mentioned problems, the present inventors succeeded in obtaining a monoclonal antibody that has specificity for ERG, and by using this monoclonal antibody, it is possible to specifically target HRG. The inventors have discovered that it is possible to easily measure this, and have completed the present invention.

The monoclonal antibody of the present invention is produced by the so-called cell fusion method. That is, it is produced by forming a fusion hybridoma between an antibody-producing cell and a myeloma cell, cloning the hybridoma, and selecting a clone that produces an antibody specific to HRG. Antibody-producing cells can be used, for example, 3977 cells derived from lymph node cells or 3977 cells derived from animals immunized with E (RG). Examples of animals to be immunized include mice, rats,
An example is a rabbit.

HRG derived from human blood can be used as an antigen;
For example, the human HRG purified from human blood plasma by heparin affinity chromatography and ion exchange chromatography is mixed with a 70% Indian adjuvant and immunized into animals. Immunization is performed by subcutaneously, intramuscularly, or intraperitoneally injecting the animal, and about 2 to 5 days after the final immunization, antibody-producing cells are collected from the immunized animal.

As myeloma cells, those derived from mice, rats, humans, etc. are used. For example, G. cell fusion.

Keller (G, K5hler) et al., Nature (
Nature) Volume 256, Page 495 (1975)
It is carried out by the method described in or a method similar thereto.

At this time, 30-50% polyethylene glycol (molecular weight 1
000-4000) at a temperature of 30-40°C.
This is carried out by reacting for about 5 minutes.

Hybridomas obtained by cell fusion are subjected to screening. That is, screening is performed by 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 that produce the desired monoclonal antibody, for example, by an enzyme antibody method. The selected clone is, for example, pristane (
2,6,10,14-tetramethylpentadecane) was intraperitoneally transplanted into BALB/C mice, and
Four days later, ascites fluid containing a high concentration of monoclonal antibodies is collected. Recovery of monoclonal antibodies from this ascites
This can be easily achieved by applying conventionally known purification methods such as ammonium sulfate fractionation, ion exchange chromatography, gel chromatography, and affinity chromatography.

The anti-HRG monoclonal antibody thus obtained can be used in biological samples such as serum, blood plasma, or urine.
It is extremely suitable for specifically and easily measuring. For this determination, it is possible to use the monoclonal antibodies themselves or 7 fragments thereof with corresponding immunological properties, for example F(a'b)t fragments.

〔Example〕

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 Preparation of monoclonal antibodies (1) Purification of antigen Polyprene (s
om9), benzamidine (final concentration 1mM), soybean trigsin inhibitor (50~), PMSF (phenylmethanesulfonyl fluoride, final concentration 11mM)
) and aprotinin (36,000 kallikrein inhibitory units), followed by the gradual addition of IM BaC480-. After addition, let stand for 1 hour, centrifuge at 7800Xg for 20 minutes to obtain a supernatant, add Q and IMEDTA solution (11mM) to this, and obtain a 35% to 8% ammonium sulfate saturated fraction by the usual method. .Redissolve this in distilled water and (
Dialyze overnight against Q:02M phosphate buffer (pH&5) 501 containing L4M NaCt. Next, the dialyzed fluid was added to a Shitaheharin-agarose column (6,8α x 116n) equilibrated with the above buffer, and I M N
Contains hCl (102M phosphate buffer (pH 45)
After elution with 1M to 5M NaCt, the adsorbed protein is eluted using a concentration gradient method of 1M to 5M NaCt. A bimodal peak is obtained and the internally eluted peaks are collected and dialyzed against 50mM TriB-HCt paao containing 10mM sodium citrate and 20mM NaCl. DlleA equilibrated with the same buffer as above.
R-Sephadex A-50 column (2,5 cm x
20 cm ) and 20–520 mM NaC
Elute the adsorbed protein using the concentration gradient method in step 1.

The peaks eluted first were collected to obtain purified HRG, which was used as an antigen.

(2) Immunization of mice Dissolve HR () in 10mM phosphate buffer (pH 7,4) containing α15M NaC2, mix well with 70 India complete adjuvant at a ratio of 1:1 (v/v), Each mouse was immunized intraperitoneally with 501 it of 5HRG. HRC 21 days after the first immunization)
50 μt was mixed with 70 India incomplete adjuvant and administered intraperitoneally, and 14 days later, HRG 20 μt
T was administered through the caudal vein.

(3) Cell fusion and cloning Three days after the final immunization, the lungs of the mice were removed,
Cells and mouse myeloma P! 1U1 at a ratio of 10=1, and cell fusion was performed according to the method of Keller et al. described in Nature. Next, it was implanted into a 96-well microplate and HA'I' (hypoxanthine I
0-' M, aminopterin 4 X l O-' M,
DMEM-10% containing thymidine 1.6X+05M)
After culturing in FC8 medium (HAT medium) for 10 to 17 days, H
DMEM-1 containing T (hypoxanthine 1 x 10-'M, f midine 1.6X + 0-sM)
Transfer to 0% FCB medium (HT medium) and further add flask (
DMEM-1 after being able to culture at 25 m)
Culture was continued in 0% FC8 medium. The antibody titer in the culture supernatant of wells in which proliferation was observed was measured by enzyme antibody method, and the desired hybridoma was cloned from appropriate wells by limiting dilution method. That is, approximately 2.5 x 10' mouse thymocytes were seeded per well in a microplate, and then 5.1, α5 cells/(Ll
-) The hybridoma was diluted and cultured by inoculating each well of IILl into the above-mentioned microplate. Colonies that were visible to the naked eye were formed 10 to 14 days after the start of culture, and a clone strain was obtained.

(4) Screening method The culture supernatant of the wells in which hybridomas and clones have proliferated is separated and treated with enzyme-linked immunosorbent.
Assay (Enzyme Linked Immunos)
Orbent As5ay) (ELI SA)
The ability to produce antibodies against HRG was investigated by a method. Dispense 0.5 μg of HRG into a microtiter plate at 150 μt/well and incubate at 4°C for 18 hours.
was adsorbed onto the solid phase. 10mM phosphate buffered saline (
After washing each well in triplicate with 200 μt of pBs) (pH 74), PBS containing 1% bovine serum albumin (BAA).
was added at 100 pL/well and allowed to stand at 57°C for 1 hour to block the unadsorbed portion of each well. Next, 50 μt/well of the culture solution as a specimen was added and reacted at 37° C. for 1 hour. After washing 5 times with PH1, 50 At/well of peroxidase-labeled anti-mouse IgG (manufactured by Cavell) diluted 1000 times was added, and the mixture was reacted at 37°C for 1 hour. Wash with PH1, Q, 0ml 1% hydrogen peroxide, α55
α1M citrate buffer (pH
4,0) was added, and the absorbance at a wavelength of 405 nm was measured. Strong color development was observed when antibodies against HRG were present in the sample.

As a result, clone strains HR-1 and H with high antibody production ability were found.
R-26 was obtained.

The clone strains are each Hybridoma HR-1
FEBM P-1
0171), designated as Hy'brloma HR-26 and published as FERM No. 10172 (FERM, P-101).
72) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

(5) Preparation of monoclonal antibodies Pristane (manufactured by Aldrich) α5- was intraperitoneally administered to BALB/Q mice aged 7 weeks or older, and after 1 week or more had passed, the clones were cultured and expanded. '
mice/mouse were inoculated intraperitoneally. After 10-14 days, mice were sacrificed and ascites fluid was collected. This at 5,000rpmlO
The mixture was centrifuged for a minute to obtain ascites containing monoclonal antibodies at 5 to 1 stnt/mouse.

(6) Purification of monoclonal antibodies The ascites obtained in (5) above was filtered through absorbent cotton to remove fat, and then added to 50 mM phosphate buffer (pa
After diluting the mixture twice with Zs), an equal volume of 10 rho saturated ammonium sulfate was added and the precipitate fraction was collected. This fraction was dissolved in as small a volume as possible of the above phosphate buffer and dialyzed against the same buffer. This sample was applied to a DEAE-cellulose column, and anti-HRG monoclonal antibody HR
-1 and HR-26 were obtained.

(7) Physicochemical properties of monoclonal antibodies■ Engineering subclass HR-1: IgG.

HR-26: IgG1 The specific class of the purified monoclonal antibodies was determined in an Ophthalony gel diffusion test using class-specific anti-mouse egg antibodies.

■ Molecular weight HR-26: 170,000±5,
000HR1, + 165,000±5,0008D
The molecular weight of the monoclonal antibody was estimated by S-polyacrylamide gel electrophoresis (12.5%). As a molecular lid marker, Bio-Rad's 5DS-PAGE molecular weight standard-HLgh was used (myosin 200kD
a, β-galactosidase ++6kDa, phospholipase 1) 97kDa, serum albumin 66kDa,
ovalbumin (4 s kDa).

■ Reactivity with antigen HR-+: CNBr 50kDa fragment! ? R-
26: Recognizing the three-dimensional structure and decomposing HRG with CNBr (T, Koide (T).

Kolθ et al., Journal of Biochemistry (J, Biochem, ) Vol. 98, No. 119
1-1200 (1985) I, dissolved in 50mM Tris-HCt buffer pHa6, and subjected to 8D8-polyacrylamide electrophoresis. Burne
tte), Analytical Biochemistry (An
al, Biochem, ) Volume 112, No. 195~
20!1 (1981)] reactivity with monoclonal antibodies was investigated.

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

■ Cross-reactivity HR-1: Does not show cross-reactivity with antithrombin III, plasminogen, and fibrinogen HR-26: Does not show cross-reactivity with antithrombin III, plasminogen, and fibrinogen Fibrinogen was dissolved in PBS to a concentration of 10 .mu.2/-, each of which was coated on a microtiter plate, and the cross-reactivity with the above substances was examined in the same manner as the hybridoma screening method.

■ Influence of coexisting substances HR-+: Reactivity with HR() is not affected by the presence of HR-26, plasminogen, fibrinogen, heparin HR-26: Presence of HR-1, plasminogen, fibrinogen, heparin 50 μt/well of HRG (dissolved in PBS to give a concentration of 5 μf/−) was added to a microtiter plate and coated at 57°C for 3 hours. After washing with PBS,
200μt/PBS containing % bovine serum albumin
Add to the wells, block for 1 hour at 37°C, and wash with PBS. In addition to this, HR-26 (10μf/-), HR-1
(10μf/-), plasminogen (10μr/rn
t), fibrinogen (10 μr/1 nt), and heparin (I IU/d) were added at 50 μt/well.
℃ for 1 hour, followed by Nakane
) law [P, ni, Nakane (P, ni, Nakane)
, Journal Op Histochemistry and Cytochemistry (J, Hlstochem, Cyt.
ochem) Volume 22, Page 1084 (1974))
50 μt/well of each prepared peroxidase (POD)-labeled HR-1 or ) (R-26) was added, reacted for 1 hour at 37°C, and washed with PBS. The absorbance at 405 nm was measured and compared with the case where no coexisting substance was added to examine the influence of the coexisting substance.

The above results are summarized in Tables 1 and 2.

Table 1 summarizes the Ig subclasses of HR-1 and HR-26, reactivity with antigens, and cross-reactivity, and Table 2 summarizes the effects of coexisting substances.
-26 each recognize different epitopes on HRG, and HRG on plasminogen, fibrinogen, and heparin.
It was shown that its reactivity was not affected by binding to and coexistence with. In particular, HR-26 was shown to recognize the three-dimensional structure of 5HRG, as its reactivity disappeared or significantly decreased upon CNBr fragmentation.

Table 1 HR-1 gG1 + (50kDa fragment) HR-26 gGl ± Antithrombin III, plasminogen, fibrinogen Table 2 Plasminogen (10μ/, j) Fibrinogen (10μf/rrt) Heparin (I
I-) HR-1 (10μf//Rt) HR-26 (10μ2/-) No L5D α50 α48 α01 α55 1.00 1.01 1.00 1.11 α01 1.01 Example 2 Using monoclonal antibody Ita Sand Inch E
Measurement of HRG by IA method Monoclonal antibodies HR-1 and HR-2 obtained in Example 1
HRf) measurement reagent was prepared using 6.

(1) Preparation of peroxidase (POD)-labeled monoclonal antibody Monoclonal antibody HR-1 was POD-labeled using the Nakane method (supra) K.

(2) Sandwich E, IA method measurement system Monoclonal antibody HR-26 (101197m
1) was added in an amount of 10 tlt and incubated overnight at 4°C. After discarding the solution, 200 tlt of a PBS solution containing 1% BSA was added to each well, and blocking was performed at 37°C for 1 hour. After washing thoroughly with PBS,
Add 20 μt of sample followed by P containing 1% BAA
After adding 100 µt of POD-labeled HR-1 antibody solution diluted with BS and reacting at 37°C for 30 minutes, washing three times with PBS, α001% hydrogen peroxide, Q, 55 ~/d AB
Q containing 'r8, 1 M citric acid-sodium hydroxide buffer (pH 4,0) was added, and the absorbance at a wavelength of 405 nm was measured.

(3) Measurement of specimen Blood samples obtained from 18 healthy subjects, 18 patients with myocardial infarction, and 12 patients with cerebral infarction were diluted 1000 times with PB8 containing 1% BSA and measured using the EIA method described above. At this time, the HRG of each sample was determined from the calibration curve obtained using purified HRG as a standard.
#Determined degree. The results are shown in Figure 1. That is, FIG. 1 shows sandwich EI using the monoclonal antibody of the present invention.
The blood plasma HRG measurement results by method A are expressed as HR() concentration (μy/lIt, vertical axis) and each side sputum sample (horizontal axis).
FIG.

In the healthy group, the average SD was 100.6 ± 1915 μf/,
It was t. On the other hand, the myocardial infarction group and the cerebral infarction group each had 11
5.2±51.3μf/lnt, 11.7.7±6
It showed a wider distribution of 1.6 μf/- compared to healthy people, and when the cutoff value was set to the average ±28D of the healthy group, some cases showed abnormal values.

Example 5 Measurement of HR() by latex agglutination method using monoclonal antibodies Monoclonal antibodies HR-1 and H'R- obtained in Example 1
HR() measurement reagent was prepared using No. 26.

(1) Sensitization of latex αI M 105M glycine-NaO containing NaCt
1 volume of a suspension of latex [manufactured by Sekisui Kagaku Kogyo Co., Ltd., particle size 0.464 μm] dispersed in H buffer (GB% pHa5) so that the solid content was 4.0% (vr/v). E (R-1 or HR-215 (30/- each)
and 1 volume of each solution were mixed and incubated at 37°C for 3 hours. This includes BSA, sucrose, choline chloride, and NaN3.
6 volumes of GB were added to give a final concentration of 0.5% latex solids, 1% BAA, 1% sucrose, 1M choline chloride, 1 ci NaN. Latexes separately sensitized with HR-1 and HR-26 were mixed at a ratio of 1:1 before use, and this was used as a latex reagent.

(2) Specimen measurement method Specimen 20/41 diluted blood plasma sample 1000 times with PBS containing 1% BSA and 20μt latex reagent
were mixed on a latex agglutination determination plate, and the presence or absence of agglutination was determined after 5 minutes. At this time, the detection sensitivity of this system using purified HRG was determined to be +50nj/-. As a result of measuring blood plasma samples of 14 healthy subjects, 13 cases of cerebral infarction, and 10 cases of myocardial infarction using this measurement method, as shown in Table 5, blood HRG was high in patients with cerebral infarction and myocardial infarction. It was shown that

Table HRC 200nf/1nt ◎ HRC 150nf/-◎ E(RG 100nf/en/ / ◎ / [Effects of the Invention] As described in detail above, the present invention provides a monoclonal antibody against 9 HRC) . By using the monoclonal antibody of the present invention, it is possible to measure HRG easily and with high specificity, and it is very useful for studying the physiological effects of HRG and diagnosing diseases related to blood coagulation and fibrinolysis. .

[Brief explanation of the drawing]

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

Claims (1)

[Claims] 1. Anti-histidine-rich glycoprotein monoclonal antibody. 2. The monoclonal antibody according to claim 1, wherein the monoclonal antibody has the following properties. (a) Molecular weight: 170,000±5,000 (b) Ig
Subclass: IgG_1 (c) Reactivity with antigen: Recognizes the three-dimensional structure of histidine-rich glycoprotein, does not cross-react with antithrombin III, plasminogen, and fibrinogen, and does not cross-react with plasminogen, fibrinogen, and heparin. 3. The monoclonal antibody according to claim 1, wherein binding to rich glycoprotein does not inhibit the reactivity of the antibody to histidine-rich glycoprotein. 3. The monoclonal antibody has the following properties. (a) Molecular weight: 165,000±5,000 (b) Ig
Subclass: IgG_1 (c) Reactivity with antigen: Reacts with a 50 kDa peptide on the N-terminal side of histidine-rich glycoprotein obtained by CNBr decomposition of histidine-rich glycoprotein, and reacts with antithrombin III, plasminogen, Does not cross-react with fibrinogen, and the binding of plasminogen, fibrinogen, and heparin to histidine-rich glycoproteins does not inhibit the reactivity of antibodies to histidine-rich glycoproteins4 Histidine-rich in biological samples - A method for measuring histidine-rich glycoprotein, which comprises using the monoclonal antibody according to claim 1 in measuring glycoprotein.