JPH0753756B2 - Anti-heparan sulfate monoclonal antibody and method for detecting heparan sulfate using the same - Google Patents

Description

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

[Prior art and its problems] Heparan sulfate (hereinafter referred to as "HS") is present on the surface of cells of almost all kinds of animals (Biochemistry, 10 ,
1445 (1971)), which is said to be the glycosaminoglycan most basically involved in the interaction between cells and cell-cell matrix.

It is also said that changes in HS are involved in the decrease in cell adhesion ability due to canceration, the increase in cell mobility, and the loss of proliferation adhesion inhibition ability.

In addition, HS is (a) involved in the regulation of DNA expression as a cell nuclear component (b) is a major proteoglycan in the nervous system (c) a lipoprotein lipase binding site as a component on the surface of vascular endothelial cells, It is thought to have various functions involved in metabolism (d) controlling cell growth (e) controlling permeability of proteins as a component of basement membrane.

As described above, HS, which is widely present in the animal body and has various functions, is an important component for animals and humans, and its qualitative or quantitative, highly specific detection is very important for biology and medicine. Have important significance to. In this sense
The HS monoclonal antibody can be a unique detection reagent.

Several reports on antibodies to proteoheparan sulfate [Proc. NAS, 77 , 4494 (1980); J. Cell. Biol., 9
9 , 1743 (1984); J. Cell. Biol., 101 , 976 (1985)].

However, these antibodies recognize the core protein of proteoheparan sulfate and do not recognize the HS sugar chain portion.

Therefore, the inventors of the present invention have conducted intensive studies for the purpose of obtaining a monoclonal antibody having excellent specificity for the HS sugar chain portion, and as a result, completed the present invention.

[Structure of the Invention] The present invention provides an anti-HS mono-produced from a hybridoma formed by cell fusion of rat splenocytes and mouse myeloma cells pre-immunized with HS-containing tissue-derived proteoheparan sulfate as an antigen. The present invention relates to a clone antibody and a method for detecting HS characterized by using the monoclonal antibody.

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 proteoheparan sulfate used as an antigen, a tissue containing HS, for example, mouse Engelb
reth-Holm-Swarm tumor (hereinafter referred to as "EHS tumor"), bovine liver, porcine liver, bovine lung, and porcine lung may be any, but particularly those derived from tissues rich in HS, For example, it is preferable to use proteoheparan sulfate derived from EHS tumor tissue.

Rats are used as the immunized animals, and among them, Wi
It is preferable to use star rats.

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 spleen is removed and the resulting lymphocytes are subjected to cell fusion. On the other hand, the tumor cell line used for cell fusion is usually P ₃ -N derived from mouse BALB / c.
S-1 / 1-Ag4-1, P ₃ -X63-Ag8-U1 (P ₃ U1), P ₃ -X63-Ag8-653, SP2
/ 0-Ag14 etc. 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)
r) or the commonly used limiting dilution method. Cloning is performed at a cell concentration such that a colony is formed from one hybridoma. In the limiting dilution method
Perform so that there are no more than one cell per well in a 96-well plate. Whichever method is used, cloning is repeated twice to obtain a single clone.

Production of Anti-HS Monoclonal Antibodies Once clones are established, antibodies are produced in large quantities either in vitro or in vivo. i
Antibodies produced in vitro do not have any contamination with other antibodies, but have low antibody titers. The antibody produced in vivo is mixed with the host a little, but the antibody titer is much higher than that in vitro. Which method is used to produce the antibody depends on the purpose.

An anti-HS monoclonal antibody can be obtained by the above method.

Detection of HS In order to specifically detect HS using an anti-HS monoclonal antibody, an enzyme antibody method, a fluorescent antibody method, a heavy metal antibody method, a biotin-avidin system, a peroxidase-antiperoxidase method, etc. can be performed according to a conventional method. Be seen.

EFFECTS OF THE INVENTION According to the present invention, a monoclonal antibody that recognizes only the polysaccharide chain of HS can be provided, and by using this, HS can be specifically detected.

[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 Proteoheparan Sulfate EHS tumor was transplanted to a mouse to grow, and this was excised. From this tumor, the method of JR Hassell et al. (Proc.Natl.A
Proteoheparan sulfate was purified and collected according to cad.Sci.USA, 77 , 4494 (1980).

(2) Preparation of antibody A part of the proteoheparan sulfate obtained in (1) was reduced with dithiothreitol. The treated and untreated ones were sensitized by combining the same amounts with 260 μg of proteoheparan sulfate and Freund's complete adjuvant, intraperitoneally and subcutaneously on the back of Slc Wistar rats. Then, booster injections were performed every two weeks for a total of five times. At this time, a mixture of reduced and untreated proteoheparan sulfate was used.
μg of proteoheparan sulfate was administered with Freund's incomplete adjuvant. After sensitization, the antibody titer was checked by an ELISA method and found to be 30,000 or more. Therefore, the rat was killed and the spleen was excised, to obtain floating splenocytes by a conventional method.
To 10 cells thus obtained, mouse myeloma NS-1
Cell fusion was performed using polyethylene glycol at a ratio of 1 cell. Then, in order to separate the hybridoma from the myeloma cells, the cells were cultured in HAT medium for 2 weeks to kill the residual myeloma cells.

(3) Cloning Cloning was performed according to a conventional method, and the antibody produced by the obtained clone was screened according to the ELISA method. At this time, clones that react with the antigen proteoheparan sulfate but not with heparitinase-treated proteoheparan sulfate were selected and two clones were obtained, named HK-144 and HK-249, respectively. These two
Proliferation of clonal cells of the species was performed to collect antibodies (hereinafter,
Monoclonal antibody derived from HK-144 is "HK-144 antibody", HK-2
The monoclonal antibody derived from 49 is called "HK-249 antibody". ).

Both antibodies were analyzed using a monoclonal typing kit of Miles Laboratories, Inc. (hereinafter referred to as "Miles"), and were identified as class IgM.

Example 2 The properties of the HK-144 antibody and HK-249 antibody obtained in Example 1 were examined. In addition, these properties are determined by the ELISA method [A.
BB, 207 , 399 (1981)] and ELISA-inhibition experiments.

(1) Measurement of antibody titer EHS Tumor-derived proteoheparan sulfate was dissolved in Voller's buffer at a protein concentration of 1 μg / ml, and 100 μl of an antigen solution was subjected to ELISA.
It was added to each well of a microtiter plate (96 wells, made of polyvinyl) for use and allowed to stand at 4 ° C for 2-3 days to coat the antigen. Plate to PBS (phosphate buffered saline) ~ 0.05% T
After washing three times with a ween20 solution, each hybridoma culture supernatant (repeated 3-fold dilution) RPMI 1640 medium, 10% FCS) was added and reacted at room temperature for 1 to 2 hours. After washing three times with PBS to Tween20 solution, peroxidase-conjugated anti-rat (IgG + IgM) rabbit immunoglobulin (DAKO) / PBS
˜Tween 20 solution (100 μl / well) was added and reacted for 1 hour at room temperature.

After washing 4 times with PBS to Tween20 solution, orthophenylenediamine to hydrogen peroxide solution [orthophenylenediamine
10 mg / 1 ml methanol, 30% hydrogen peroxide aqueous solution 10 μl, distilled water 100 ml] 150 μl was added to develop color, and after stopping the reaction with 3 M sulfuric acid 50 μl, the absorbance at 480 nm was measured.

Thus, the antibody titers of the HK-144 and HK-249 antibody solutions were measured. The results are shown in FIG.

An antibody titer of about 700 times or more was observed for both antibodies.

(2) Effect of enzymatic digestion on proteoheparan sulfate (A) Heparitinase (heparan sulfate degrading enzyme) EHS Tumor-derived proteoheparan sulfate (protein 1 μg / m
l, uronic acid 0.5 μg / ml) solution 100 μl / well, and then washed 3 times with PBS to Tween 20 solution. Heparitinase
After digestion with 0.2mIU / well at 37 ℃ for 30 minutes, PBS-Tween
After washing twice with 20 solutions and removing the enzyme, 100 μl / well of each antibody was added and reacted. As a control, a monoclonal antibody HK-102 that recognizes the core protein part of proteoheparan sulfate.
Contrasted with. The results are shown in FIG. In FIG. 2, (−) represents the result when heparitinase was not treated, and (+) represents the result when heparitinase was treated.

Since HK-144 antibody and HK-249 antibody do not show binding to proteoheparan sulfate treated with heparitinase, it can be seen that they are sugar chain-recognizing antibodies.

(B) Pronase (proteolytic enzyme) 100 μg of proteoheparan sulfate was dissolved in 200 μl of water, and pronase (10 mg / ml 1M Tris-HCl buffer pH 7.4) was added to
8 μl was added and reacted at 50 ° C. for 24 hours. 1 minute for 5 minutes
After heating at 00 ° C. to inactivate the enzyme, it was precipitated with ethanol, the supernatant was removed, dried and dissolved in 100 μl of PBS-Tween 20 solution. As a control, inactivated pronase was added to proteoheparan sulfate, and immediately ethanol-precipitated was used. These solutions are serially diluted to produce HK-144 and HK-2.
49 As a result of the inhibition experiment against each antibody, it was found that the inhibitory activity of proteoheparan sulfate was hardly changed even when digested with pronase (results omitted). This is a result of supporting that both antibodies recognize sugar chains.

(3) Specificity of HK-144 antibody and HK-249 antibody In order to examine the reaction specificity of the monoclonal antibody of the present invention for various sugars, an inhibition experiment by an ELISA method was performed.

Each well was coated with 100 μl of EHS tumor-derived proteoheparan sulfate (protein 1 μg / ml) solution, and then washed 3 times with PBS-Tween 20 solution. The HK-144 and HK-249 antibody solutions were used at 40 μl / well. Various glycosaminoglycans for investigating the reaction specificity of the antibody are each glycosaminoglycan 1 mg /
Using ml stock solution as a starting solution, prepare a serial dilution solution,
80 μl of each concentration of glycosaminoglycan solution and each antibody solution were simultaneously added to wells coated with proteoheparan sulfate, and allowed to bind with antigen-antibody at room temperature for 1 to 2 hours. PBS
~ Anti-rat (IgG + IgM) rabbit immunoglobulin / PBS conjugated with peroxidase after washing with Tween20 solution ~
100 μl / well of Tween 20 solution was added and reacted at room temperature for 1 hour. Unreacted substances were washed, 150 μl of ortho-phenylenediamine-hydrogen peroxide aqueous solution was added, and the color tone generated by the reaction was examined by measuring the absorbance at 480 nm.

The absorbance corresponding to the added amount of each glycosaminoglycan was plotted, and the added amount corresponding to 1/2 value of the absorbance when no inhibitor was added was defined as the 50% inhibition amount (IC ₅₀ ). The results are shown in Table 1. In Table 1, the symbol> indicates that inhibition does not occur at least up to this amount.

From Table 1, the monoclonal antibody of the present invention does not react with glycosaminoglycans other than HS, and the HK-144 antibody has a broad specificity of reacting with HS derived from various animals. It can be seen that the 249 antibody has a narrow specificity that reacts only with mouse EHS tumor-derived HS.

From the above results, anti-HS monoclonal antibody of the present invention, HS
It can be said that the present invention provides a useful and convenient method for diagnosing various diseases, particularly malignant tumors, since it specifically recognizes only the polysaccharide chain.

(4) Specificity of HK-144 antibody and HK-249 antibody (reactivity study by chemical modification of HS) (A) Acetylation of —NH ₂ group 50 μg of HS derived from EHS tumor was dissolved in 80 μl of water and saturated NaHCO 3 was added.
₃ Aqueous solution (20 μl) and 5% (w / v) acetic anhydride aqueous solution (20 μl) were added and stirred, and then allowed to stand at room temperature for 10 minutes. After ethanol precipitation was repeated twice, it was dried.

(B) Desulfation of N-SO ₃ group The method of Nagasawa et al. [Carbohydrate Research, 46 , 87 (197
6)], the N—SO ₃ group was desulfated.

50 μg of EHS tumor-derived heparan sulfate was dissolved in 80 μl of water, treated with Dowex 50W (H ⁺ ), converted to H ⁺ form, and neutralized with pyridine to give a pyridine salt. After drying, it was dissolved in 100 μl of dimethyl sulfoxide containing 5% of water, reacted at 50 ° C. for 90 minutes, and neutralized with 0.2N NaOH aqueous solution. Ethanol precipitation 2
After repeating the times, it was dried.

(C) Control EHS Tumor-derived HS was dissolved in 80 μl of water to serve as a control.

To each of the samples prepared in (A), (B), and (C), 200 μl of the monoclonal antibody solution of the present invention was added, and added to wells previously coated with EHS tumor-derived proteoheparan sulfate.
An inhibition experiment was performed by the ELISA method. The results are shown in FIG.

In the case of the HK-144 antibody, the specificity was not changed by the chemical modification of HS. On the other hand, in the case of the HK-249 antibody, no change due to acetylation was observed, but the inhibitory activity against the antibody disappeared due to desulfation of the N-SO ₃ group.

[Brief description of drawings]

FIG. 1 is a diagram showing the measurement results of antibody titers. FIG. 2 is a diagram showing test results for the effect of heparitinase digestion on the reactivity of the monoclonal antibody of the present invention. FIG. 3 is a diagram showing test results on the effect of chemical modification of HS on the reactivity of 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) (56) References The Journal of Clinical Investigation, 77 (6) (1986) P.M. 1824-1830

Claims (8)

[Claims] 1. A monoclonal antibody produced from a hybridoma formed by cell fusion between rat spleen cells and mouse myeloma cells pre-immunized with heptane sulfate-containing tissue-derived proteoheparan sulfate as an antigen. , Bound to heparan sulfate derived from mouse Engelbreth-Holm-Swarm tumor tissue, chondroitin-4-
An anti-heparan sulfate monoclonal antibody that does not substantially bind to any of sulfate, chondroitin-6-sulfate, hyaluronic acid, and heparan sulfate derived from normal tissues of bovine kidney. 2. A tissue containing heparan sulfate is
The monoclonal antibody according to claim 1, which is an Engelbreth-Holm-Swarm tumor tissue. 3. The monoclonal antibody according to claim 1, which binds to heparan sulfate derived from normal tissues of mouse lung and kidney and bovine lung. 4. The monoclonal antibody according to claim 1, which does not bind to heparan sulfate derived from normal tissues of mouse lung and kidney and bovine lung. 5. A monoclonal antibody produced from a hybridoma formed by cell fusion between rat spleen cells and mouse myeloma cells pre-immunized with heptane sulfate-containing tissue-derived proteoheparan sulfate as an antigen. , Bound to heparan sulfate derived from mouse Engelbreth-Holm-Swarm tumor tissue, chondroitin-4-
A method for detecting heparan sulfate, which comprises using an anti-heparan sulfate monoclonal antibody that does not substantially bind to any of sulfate, chondroitin-6-sulfate, hyaluronic acid, and heparan sulfate derived from normal tissues of bovine kidney. . 6. A tissue containing heparan sulfate is
The detection method according to claim 5, which is an Engelbreth-Holm-Swarm tumor tissue. 7. The method according to claim 5, wherein the anti-heparan sulfate monoclonal antibody binds to heparan sulfate derived from normal tissues of mouse lung and kidney and bovine lung. 8. The detection method according to claim 5, wherein the anti-heparan sulfate monoclonal antibody does not bind to heparan sulfate derived from normal tissues of mouse lung and kidney and bovine lung.