PREPARATION AND SPECIFICITY OF ANTIBODIES TO AN ANTI-TUMOR ~-GLUCAN, LENTINAN

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1 Vol. 39, No. 4, July 1996 Pages PREPARATION AND SPECIFICITY OF ANTIBODIES TO AN ANTI-TUMOR ~-GLUCAN, LENTINAN Masashi MIZONO 1, Ken-ichiro MINATO l and Hironobu TSUCHIDA l 1The Graduate School of Science and Technology, Kobe University, Nada-ku, Kobe 657, Japan Received April 9, 1996 SUMMARY: Antibodies against ~-glucan, lentinan from "Shiitake" (Lentinus edodes), were raised in the rabbit by subcutaneous immunization. Our antibodies reacted significantly with lentinan by inhibition assay of ELISA. The antibodies did not recognize the other polysaccharides such as amylose, dextran, laminarin and galactan. It was proved that lentinan contents in mushroom could be measured by ELISA with the anti-lentinan antisera. Its contents were 3.5 mg/g fresh weight in Lentinus edodes. However, lentinan was not contained in Agaricus brazei, Agaricus bisporus and Rwnaria bitrytis. Key words: Lentinan, anti-tumor activity, polysaccharides, ELISA, polyclonal antibody, Lentinus edodes (Shiitake) Lentinan is a polysaccharide isolated from the water-soluble extracts of "Shiitake" (Lentinus edodes) composed of a backbone of ~-(1-*3)-linked-D-glucose residues. This ~-(1-*3)-D-glucan is possessed of two [~-(1 ~6)glucose side chains of ~- D-glucosyl groups for every five glucose residues (1). Recently, it has been reported that lentinan had strong host-mediate anti-tumor activities against various tumors. In addition, the combination therapy with lentinan and another chemotherapeutic agent had marked effects on tumors. The combination therapy with lentinan and ril-s, T cell growth factor, had prolonged the life of mice bearing Madison lung carcinoma, and inhibited the pulmonary metastasis of it (2). In the presence of monoclonal antibody against human gastrointestinal tract tumors, lentinan had also enhanced the cytotoxic and cytostatic activities of murine peritoneal macrophages against the tumor cells (3). These studies may provide the approaches to immunotherapy to various tumors. Although there are some reports that lentinan had /96/ $05.0(3/0 Copyright 1996 by Academic Press Australia. All rights of reproduction in any form reserved.

2 antitumor activities, little has been reported what contents of lentinan were contained in mushrooms. Because it is so difficult to purify and quantify easily lentinan in the mushroom. Immunoassay seems to be a simple and exact method of analysis of the glucan (4, 5). Tabata et al. reported about the preparation of polyclonal antibodies to Schizophyllan (6). Recently, Hiram et al. reported that they had succeeded in preparing monoclonal antibodies against Schizophyllan, ~-glucan, in Schizophyllum commune (7). We planned to make use of enzyme-linked immunosorbent assay (ELISA) to quantify easily lentinan contents in extracts from some mushrooms. This paper describes the preparation of polyclonal antibodies and the specificity to lentinan compared with the other polysaccharides. Moreover, the contents of lentinan or lentinan-like polysaccharides in Lentinus edodes or the other mushrooms are measured by inhibition assay of ELISA using the anti-lentinan antisera. MATERIALS AND METHODS A nimals. New Zealand White rabbit (female, 16 weeks of ages) was purchased from Japan SLC Co. (Shizuoka, Japan). Materials. Purified lentinan was kindly gifted by Ajinomoto Co. Laminarin (13-1,3-glucan), Dextran (tx-l,6-glucan, M.W. 1.1 x 104) were purchased from Sigma Chemical (St. Louis, MO). Pullulan (a-l,6-glucan, M.W x 104) was purchased from Showa Denko Co. (Tokyo, Japan). Galactan extracted from Gum Arabic was purchased from Aldrich Co. (Milwaukee, WI). Amylose (~x-l,4-glucan, M.W. 2.9 x 104) was purchased from Nacalai Tesque Co. (Kyoto, Japan). Mushrooms. Lentinan edodes cultivated in Ohya, Hyogo was used. Maripilus giganteus, Panellus serotinus, Sarcodon aspratus and LactaHus hatsudake were collected at the seminar forest of Mie University. Agaricus brasei was kindly provided by Iwade Mushroom Institute, Mie. Agadcus bisporus and Flammulina velutipes were purchased from a local market. Rconaria botrytis was kindly provided by Sakekawa Agricultural Cooperative Association, Yamagata, Hygrophorus russula was collected in Yamadera, Yamagata. Grifolafrondosa was kindly provided by Sanda Mushroom Center, Nisshoku K.K., Hyogo. Preparation of anti-lentinan antibodies. Lentinan (0.5 mg) was dissolved in 1 ml of phosphate buffered saline. This solution was emulsified with an equal volume of Freund's complete adjuvant (FCA). The emulsion was injected subcutaneously at 10 different sites in the back of the rabbit. Half volume of lentinan in first dosage was boosted again two weeks after the injection. After the boost, the blood were collected to obtain anti-lentinan sera three times every one week. The sera were stored at -80 (2 until use. After measurement of the antibody titer, the rabbit were exsanguinated. Titer of the antibodies. The titers of the antisera were measured by ELISA as following. The solution (100 btl) of antigen (lentinan in PBS, 5 ~tg/ml) was placed in micro-titer wells (96 wells, Sumitomo Bakelite Co., Tokyo, Japan), and coated overnight at 4 C. Then, all wells were filled with PBS containing 1% skim milk for blocking and kept for 2 h at 20 (2. After the blocking solution was removed by decantation and washed three times with PBS-Tween (0.02% Tween 20 in PBS), 100 gl of 1:100, 1:1,000, and 1:10,000 diluted antisera was added to the micro-titer wells. The wells 680

3 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL were incubated for 1.5 h at 20~C. After washing three times with PBS-Tween solution again, 100 ].tl of 1:2000 dilute peroxidase-conjugated goat anti-rabbit IgG serum (Wako Pure Chemical Industries LTD., Osaka, Japan) was added to the wells and incubated for 1.5 h at 20~C. After frequent washing and coloration with H202-O-phenylenediamine (100 Ixl), absorbance at 492 nm was measured by ELISA Auto Reader (Corona Electric Co. LTD., Ibaragi, Japan). Inhibition assay of ELISA. Inhibition assay of ELISA was used to conf'trm the specificity of the anti-lentinan antibodies against polysaccharide-structure essentially as described by Tokano et al. (4, 5) with slight modification. The solution of antigen (lentinan in PBS, 100 [tg/ml) was coated in micro-titer wells overnight at 4~C. The antisera were incubated with the samples or various standard polysaccharides dissolved in PBS for 30 rain. After blocking ofthe wells with skim milk and washing, the solution (100 ~1) mixed with the antibodies and samples were added to the wells. The plate were incubated for 1 h at 4~C and washed with PBS-Tween. Peroxidase-conjugated goat anti-rabbit IgG serum was added, and the plate was incubated for 1.5 h at 20~C. After washing and coloration with H202-O-phenylenediamine (100 ~1), absorbance at 492 nm was measured. Lentinan contents in fresh mushrooms. Crude lentinan fractions from mushrooms were prepared essentially according to Chihara et al (8, 9) The fresh fruits bodies (20 g) of various mushrooms were homogenated with liquid nitrogen by warning blender and lyophilized. The lyophilit~ samples were exwacted with hot water (100 ml) for 8 h. The suspension were filtered to remove insoluble matters. The crude polysaccharide fractions were obtained by precipitation with equal volume of ethanol to the filtrate. The contents of lentinan and lentinan-like polysaccharides in mushrooms were measured by the inhibition assay of ELISA. RESULTS AND DISCUSSION Titer of anti.lentinan antibodies. Anti-lentinan antibodies were raised in a rabbit by immunization. The titer of the antisera as evaluated by ELISA are shown in Table i. The serum before the infection did not response against lentinan. The titer of the antisera were expressed the dilution magnification that was given an equal absorbance of non-immunization serum (Table 1). The titer was increased to maximum in two weeks after first infection. After booster dose, it kept maximum value for about one month (Fig. 1). The antibodies showed significant reaction with lentinan. As shown in Fig. 2, standard curve was calculated by the inhibition assay of ELISA. In the range of 2 to 50 ~tg/ml, the linear calibration curve was obtained. It was proved that lentinan contents could be sufficiently measured by ELISA using the anti-lentinan antisera we prepared. Specificity of anti-lentinan antisera. The specificity of the anti-lentinan antisera to the some polysaccharides was shown in Fig. 3. The antisera reacted only with lentinan but not with other polysaccharides at all. Especially, it did not react with straight chain, [~-(1, 3)-D-glucan, 681

4 Table 1. Reactivities of the rabbit anti-lentinan sera as assessed by ELISA Antisera Dilution of antisera Absorbance at 492 nm Anti-lentinan serum 1: :1, :10, [] [] D A-" ~6 ooster dose t. ~ ~4 I I / 1 I Days I I Fig. 1. Titer of anti-lentinan antisera. laminarin which composed of the main chain in lentinan. Nor this antisera recognized a-glucosebonds such as dextran and amylose. This seemed to indicate that the antilentinan antisera might just recognized mainly [~-(1, 6)-finked glucose branches repeated at regular proportion in I~-1,3 glucopyranoside main chain. The contents of lentinan in various fresh mushrooms. The contents of lentinan or lentinan-like polysaccharides in mushrooms were calculated by inhibition assay of ELISA. As shown in Table 2, the contents of lentinan in Lentinus edodes was highest and was 3.5 mg/g fresh weight (f. w.). In the other mushrooms tested, its contents were higher with successive, 2.5, 2.3, 2.2 mg/g f.w. in Flammulina velutipes, Lactarius hatsudake and Sarcodon aspratus, respectively. However, in Agaricus brazei, Agaricus bisporus and Rcunaria botrytis, lentinan-like polysaccharides were not detected. There is no relationship in the species of the tested mushrooms. As the contents of lentinan in hot-water soluble fraction from Agaticus 682

5 eq ,.Q < Fig Concentration of lentinan (pg/ml) 14! Calibration curve of lentinan by the inhibition ELISA assay. the range of a linear relationship. 1.2 "~0. lo The bar showed 0.6.< (] Concentration (~g/ml) Fig. 3. Specificity of anti-lentinan antibodies to polysaccharides. Q; Lentinan, A; Laminarin, Y; Amylose, O; Dextran, ; Galactan. 683

6 Table 2. The contents of tentinan in mushrooms as assessed by ELISA Mushrooms Contents of lentinan or lentinan-like polysaccharides (mgog 1 fresh weight) Ramaria botrytis 0 Flammulina velutipes Sarcodon aspratus MeripiIus giganteus Panellus serotinus Lactarius hatsudake O. 8 Lentinus edodes Hygrophorus russula Agaricus brazei 0 Agaricus bisporus 0 Glifora frondosa brazei (10, 11) and Gliforafrondosa (12) were very low or less, and the antitumor polysaccharides of them contained ~-(1, 3)-D-glucan as main chain, this seems that structure and molecular weight of antitumor glucan are different in the family of mushroom. ACKNOWLEDGMENT The authors thank Ajinomoto Co. Japan for giving the pure lentinan. study was supported by Iwade Mushroom Institute, Japan. This REFERENCES (1) Sasaki, T. andtakasuka, N., CarbohydrRes., 47, 99 (1976). (2) Shiio, T., Kitamura, K., Ohnishi, K. and Yugari, Y., Biotherapy, 1, 122 (1987). (3) Herlyn, D., Kaneko, Y., Powe, J., Aoki, T. and Koprowski, H., Jpn. J. Cancer Res., 76, 37 (1985). (4) Takano, Y., Yagita, H., Iida, N., Hashimoto, H., Okumura, K. and Hirose, S., Int. Arch. Allergy Appl. ImmunoL, 87, 55 (1988). (5) Takano, Y. and Okumura, K., ImmunohematoIogy, 1 0, 331 (1988). (6) Tabata, K., Itoh, W., Hirate, A,, Sugawara, L and Mori, S., Agric. Biol. Chem., 54, 1953 (1990). (7) Hirata~ A., Itoh, W., Tabata, K., Kojima, T., Itoyama, S. and Sugawara, I., Biosci. Biotech. Biochem., 5 7, 125 (1993). 684

7 (8) Chihara, G., Maeda, Y., Hamuro, J., Sasaki, T. and Fukuoka, F., Nature, 222, 687 (1969). (9) Chihara, G., Hamuro, J., Maeda, Y.Y., Arai, Y. and Fukuoka, F., Cancer Res., 30, 2776 (1970). (10) Mizuno, T., Hagiwara, T., Nakamura, T., Ito, H., Shirnura, K., Sumiya, T. and Asakura, A., Agric. Biol. Chem., 5 4, 2889 (1990). (11) Mizuno, T., Inagaki, R., Kanao, T., Hagiwara, T., Nakamura, T., Ito, H., Shimura, K., Sumiya, T. and Asakura, A., Agric. Biol. Chem., 5 4, 2897 (1990). (12) Namba, H., Hamaguchi, A. and Kuroda, H., Chem. Pharm. Bull., 35, 1162 (1987). 685