JPH0745518B2 - Anti-chondroitin sulfate D monoclonal antibody and method for detecting chondroitin sulfate D using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a monoclonal antibody having high specificity for chondroitin sulfate D and a method for detecting chondroitin sulfate D using the same.

[Prior Art and Its Problems] It is well known that chondroitin sulfate, together with other acidic mucopolysaccharides, widely exists as a substrate component of various connective tissues. This chondroitin sulfate has a disaccharide in which N-acetylgalactosamine (hereinafter referred to as “GalNAc”) and D-glucuronic acid (hereinafter referred to as “GlcUA”) as a glycoside bond as a structural unit, and sulfates at the C-4 position of GalNAc. Group-bonded chondroitin sulfate A (hereinafter referred to as "CS-A"), chondroitin sulfate C having a sulfate group bonded to the C-6 position of GalNAc (hereinafter referred to as "CS-C"), and GalNAc C.
Chondroitin sulfate D (hereinafter referred to as "CS-D") in which a sulfate group is bound to the 6-position and the C-2 position of GlcUA, respectively.
Chondroitin sulfate E (hereinafter referred to as "CS-E") having a sulfate group bonded to the C-4 position and C-6 position of GalNAc, and other L-iduronic acid (hereinafter referred to as "IdoUA") and GalNAc are glycoside bonded. , A chondroitin sulfate B having a sulfate group bonded to the C-4 position of GalNAc (also referred to as dermatan sulfate; hereinafter referred to as "CS-B"), and a sulfate group at the C-2 position of IdoUA. It is known that there is a type of structural unit such as dermatan polysulfate bound to.

Of these, CS-A and CS-C are widely found in vertebrates, and are easily available due to their high content,
Various studies have been made. CS-B is present in various connective tissues such as blood vessels, but its content is low and it is mainly present in skin tissues, and uronic acid is another chondroitin sulfate (hereinafter referred to as “CS”), which is GlcUA. In contrast, IdoUA is different. In contrast, CS-D is widely
Despite being contained in A, CS-C, and CS-E as a hybrid chain, it has been largely ignored to date because of its low content in higher animals.

In addition, a recent report that a sulfate group is bound to the C-2 position of GlcUA is an essential condition for endocytosis of heparan sulfate, which is one of the same mucopolysaccharides (NSFedarko, and HEConrad : J. Cell Biol., 102 , 58
7 (1986)), CS-D having a similar structure or a mixed chain containing CS-D has a migration and distribution of mucopolysaccharide in vivo,
It is easily assumed that they are deeply involved in metabolism and play important physiological functions. On the other hand, it is expected to play an important role as a marker for grasping the state of the tissue. That is, it has been recognized that in various diseases, mucopolysaccharides in the affected area are changed depending on the pathological condition. For example, malignant tumors are known to contain a large amount of CS-C. In this case, CS, which is a trace component contained in CS-C
Detecting and measuring -D can also detect subtle changes earlier because CS-D is a trace amount of a unique component, and is considered to have important diagnostic significance.

However, as a method for measuring CS-D, there is an oxygen chemical method in which proteochondroitin sulfate is taken out from a tissue, treated with chondroitinase, and the resulting degradation product is identified to measure CS-D. However, this method is complicated and time-consuming, and it is difficult to detect a trace amount of a component, and it is difficult to say that this method is appropriate as a diagnostic method. Therefore, the present inventors have focused on an immunological method as a method for simply and quickly measuring CS-D.

Several reports on monoclonal antibodies against CS [RB Jenkins, et al .: J. Biol. Chem., 256 , 8279 (198
1); Z.Avnur, et al.:Cell, 38 , 811 (1984)],
Of these, the monoclonal antibody of Jenkins et al.
The monoclonal antibody of Avnur et al. Recognizes not only CS-C but also CS-A and heparan sulfate, both of which are high-molecular CS-D. It does not recognize it specifically.

Therefore, the present inventors have completed the present invention as a result of intensive studies for the purpose of obtaining a monoclonal antibody having excellent specificity for CS-D.

[Structure of the Invention] The present invention provides an anti-CS produced from a hybridoma formed by cell fusion of lymphocytes of a mouse previously immunized with a proteochondroitin sulfate derived from a tissue containing CS-D as an antigen and mouse myeloma. -D monoclonal antibody and characterized by using the monoclonal antibody
The present invention relates to a CS-D detection method.

Hereinafter, the present invention will be described in more detail.

The first step in achieving the objectives of the present invention is to establish novel monoclonal hybridomas that produce antibodies. The specific details of the method for establishing this hybridoma are shown in the examples, but briefly consist of the following three steps.

1. Immunity 2. Cell fusion 3. Hybridoma selection and cloning Immunization In the present invention, as proteochondroitin sulfate used as an antigen, tissues containing CS-D, such as chicken embryo cartilage, shark cartilage, whale cartilage, bovine Any source may be used as long as it is derived from nasal cartilage or the like, but is particularly one derived from a tissue rich in CS-D, one that is easily available, and one that does not contain strong antigenic keratan sulfate, such as chicken embryo limb. Proteochondroitin sulfate derived from undifferentiated bud tissue (hereinafter referred to as "PG-
M ”. ) Is preferably used.

Conventionally, proteochondroitin sulfate obtained from these tissues was treated with hyaluronidase to remove the excess sugars that could be formed and used as an antigen having an increased protein content (Jenkins et al. Method). Uses the proteochondroitin sulfate obtained from the tissue as an antigen without enzymatic treatment.

Mice are used as the immunized animals, and among them, BALB / c, which is a well-established myeloma cell line that does not produce immunoglobulin, is preferably used.

Usually, immunization is divided into several times, but it is preferable to administer the first immunization together with an adjuvant. As the adjuvant, alum, killed tuberculosis bacteria, nucleic acid, Freund's adjuvant and the like are used.

Cell fusion After the final immunization, the lymph node or liver is removed and the resulting lymphocytes are subjected to cell fusion. On the other hand, tumor cell lines used for cell fusion are usually P3-NS-1, P3-X63-Ag8.
-U1 and SP2 / 0-Ag14 (SP2 / 0) are used.

Selection of Hybridoma and Monocloning The cell culture supernatant in which the hybridoma proliferates is selected by various analysis methods (eg, RIA, plaque method, agglutination reaction, ELISA, etc.) to select the antibody-producing hybridoma of interest. Once the hybridoma is obtained, it is cloned. FACS (Fluorescent Activated Cell Sorte)
There is a commonly used limiting dilution method in addition to the method of picking colonies using r) or Soft Agar. Cloning is performed at a cell concentration such that a colony is formed from one hybridoma. 96 by the limiting dilution method
Use a dilution such that there are about 1 cell per well in the well plate. Whichever method is used, cloning is repeated twice to obtain a single clone.

Production of Anti-CS-D Monoclonal Antibody Once a clone has been established, the antibody is produced by culturing it in large quantities in vitro or in vivo. i
Antibodies produced in vitro have no contamination with other antibodies, but have low antibody titers. Antibodies produced in vivo contain a small amount of host-derived antibodies, but the antibody titers are much higher than in vitro. Which method is used to produce the antibody depends on the purpose.

By the above method, an anti-CS-D monoclonal antibody can be obtained.

Detection of CS-D In order to specifically detect CS-D or a mixed chain containing it using an anti-CS-D monoclonal antibody, usually RIA or ELI is used.
It is carried out by SA etc. When used in ELISA, β-galactosidase, alkaline phosphatase, peroxidase or the like can be used as labeling oxygen.

[Effect of the Invention] According to the present invention, compared with the conventional product, 0.004 μg / ml or more of CS
Monoclonal antibody which specifically recognizes sensitive polymer CS including -D structural unit _{(GlcUA2-SO 4 -GalNAc6-SO} 4) can be provided, CS of tissue or body fluid by using the same
-D structural unit _{(GlcUA2-SO 4 -GalNAc6-SO} 4) can be specifically detected a polymer CS including.

[Examples of the Invention] Hereinafter, the present invention will be described in more detail with reference to Examples, but these Examples do not limit the scope of the present invention.

Example 1 (1) Preparation of PC-M After fertilized chicken eggs were incubated at 37 ° C. for 3 days, limb buds were collected. After washing with Hanks' solution, 4M guanidine hydrochloride, 2m
M Phenylmethylsulfonyl fluoride (PMSF), 5 mM E
A 50 mM Tris-HCl buffer (pH 7.4) containing DTA, 2 mM N-ethylmaleimide (NEM) and 0.36 mM pepstatin was added, and the mixture was vigorously stirred at 0 ° C. for 24 hours for extraction. At 0 ° C
Centrifuged at 10,000 rpm for 10 minutes. Cesium chloride 1.35 g / ml was added to the supernatant, and the mixture was subjected to sedimentation equilibrium centrifugation at 40,000 rpm for 2 days at 10 ° C. After pooling parts with a density of 1.35 or more, 4M guanidine hydrochloride, 2mM PMSF, 5mM EDT was used as the external solution.
Dialysis was performed using 50 mM Tris-HCl buffer (pH 7.4) containing A, 2 mM NEM and 0.036 mM pepstatin. Density gradient centrifugation (25,000 rpm, 19 ° C., 43 hours) was performed using 10% and 30% glycerin containing 4 M guanidine hydrochloride, respectively. Uronic acid was quantified and the uronic acid-containing fractions were collected.
When PG-M obtained by subjecting the fraction to ethanol precipitation was analyzed, glycosaminoglycan of PG-M used as an antigen was a mixed chain of the following components.

_{GlcUA-GalNAc4-SO 4 (CS} -A structural unit) 26.4% GlcUA-GalNAc6-SO 4 (CS-C structural units) 38.1% IdoUA-GalNAc4-SO 4 (CS-B structural units) 4.5% GlcUA2-SO ₄ - GalNAc6-SO ₄ (CS-D structural unit) 3.0% Others (chondroitin) 28.0% Core protein molecular weight about 550,000 (SDS electrophoresis) Average molecular weight of chondroitin sulfate chains about 60,000 [Bio-Gel A1. Gel permeation method using 5 m] (2) Preparation of antibody PG-M obtained in (1) was treated with Dulbecco's phosphate buffered saline (PB
S), and PG-M 50 μg / 200 μl (containing Freund's complete adjuvant) was subcutaneously administered to female BALB / c mice.
Two weeks later, as a booster, PG-M 50 μg / 200 μl
(Containing Freund's incomplete adjuvant) was subcutaneously administered twice. One week later, 1.3 × 10 ⁸ splenocytes and 2.5 × 10 ⁷ myeloma NS-1 cells were washed with RPMI1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and then centrifuged. Polyethylene glycol was added and incubated at 37 ° C for 5 minutes.
10 ml mixture of RPMI 1640 medium, fetal bovine serum and HAT medium
Was added to stop the reaction. It was diluted with a mixed solution of RPMI 1640 medium, fetal calf serum and HAT medium, and 100 μl of each was placed in a microtiter plate in which macrophages were pre-seeded as feeder cells. Incubated for 2 weeks at 37 ° C in a CO ₂ incubator. Take the supernatant of the hybridoma colonies that have developed and use ELISA.
Analysis was carried out by the method.

(3) Cloning Chicken embryo cartilage-derived proteochondroitin sulfate (hereinafter referred to as "PG
-H ". ) Is used as an antigen to assay the culture solution, and the cells (hybridomas) in the positive wells are diluted,
Incubated at 37 ° C for 1 week in a CO ₂ incubator. The colonies were similarly checked by the ELISA method, and positive wells were similarly diluted to obtain MO-225-K.

MO-22 with the Ab-ID EIA Kit A from Zymet
5-K was identified as class IgM.

Example 2 Specificity of Anti-CS-D Monoclonal Antibody MO-225-K is a monoclonal antibody obtained by using PG-M as an antigen, but not only PG-M, but also major proteochondroitin of chicken embryo cartilage. It also reacted with PG-H, which is sulfuric acid.
Furthermore, it also reacted with human cartilage proteoglycans across species.

The results of ELISA using PG-H as an antigen are shown below. E
The primary antibody in LISA was the hybridoma supernatant (RPMI 1640
A medium (10% FCS) was used, and the secondary antibody used was a goat IgG that recognizes mouse IgM (or M and G) and peroxidase bound thereto (manufactured by TAGO). PG-H is, Oike et al's method from chick embryo epiphyseal cartilage (J.Biol.Chem., 257, 9751 ( 198
2)).

(1) Binding test Each enzyme [chondroitinase AC-II (hereinafter referred to as "Chase"] was added to an ELISA microtiter plate coated with PG-H.
AC-II ". ) 0.01 unit / well, chondroitinase ABC (hereinafter referred to as “ChaseABC”) 0.01 unit / well or keratanase 0.1 unit / well], and further add hybridoma supernatant, react at 37 ° C. for 1 hour, and then at room temperature. After reacting for 2 hours, washing, secondary antibody reaction, washing, and color development were performed. As a control, HM-120 (IgG1), which is a mouse monoclonal antibody that recognizes keratan sulfate, was used. The results are shown in Fig. 1.

From FIG. 1, it can be seen that MO-225-K is bound to the portion of PG-H that is degraded by chondroitinase, that is, the chondroitin sulfate chain.

(2) Inhibition test The reaction between MO-225-K and PG-H is not inhibited at all by mucopolysaccharides other than CS, for example, various heparan sulfates, dermatan sulfates, keratan sulfates, hyaluronic acid and heparin. In other words, it shows no binding reaction with these polymers.

In addition, the same test was conducted on CS derived from various animals,
The amount required for 50% inhibition was determined. The results are shown in Table 1. In Table 1, the symbol> is 50 at least up to this amount.
It shows that it does not lead to% inhibition. This result is MO
-225-K and PG-H binding reactions are more strongly inhibited as the CS-D content increases, in other words, this monoclonal antibody specifically recognizes the D structure in the CS chain. Is shown.

From the above results, it can be said that the anti-CS-D monoclonal antibody of the present invention specifically recognizes only macromolecule CS-D, and thus provides a useful and convenient method for diagnosing various diseases.

[Brief description of drawings]

Figure 1 shows that the binding reaction between MO-225-K and PG-H was
It is a figure which shows how it is affected by enzymatically treating -H.

─────────────────────────────────────────────────── ─── 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) (56) References The Journal of Biological Chemistry, 256 [16] (1981) P.M. 8279-8282 Cell, 38 [3] (1984) P. 811-822

Claims (2)

[Claims] 1. A chondroitin sulfate chain produced from a hybridoma formed by cell fusion between a lymphocyte of a mouse and a myeloma of a mouse previously immunized with proteochondroitin sulfate derived from a tissue containing chondroitin sulfate D as an antigen. A monoclonal antibody that recognizes only the D structural unit. 2. A chondroitin sulfate chain produced from a hybridoma formed by cell fusion between a lymphocyte of a mouse and a myeloma of a mouse previously immunized with proteochondroitin sulfate derived from a tissue containing chondroitin sulfate D as an antigen. A method for detecting chondroitin sulfate D, which comprises using a monoclonal antibody that recognizes only the D structural unit.