抗五种赤潮藻的单克隆抗体的制备和特性分析
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摘要
目的：建立分泌抗海洋褐胞藻、卵圆褐胞藻、赤潮异弯藻、具齿原甲藻、尖刺拟菱形藻等五种藻的高特异性、高亲和性单克隆抗体杂交瘤细胞株。若该类细胞株所分泌的单克隆抗体结合特定藻抗原，则可利用此类单克隆抗体鉴别此五种不同的藻，从而为赤潮藻的检测、监测和防治提供有效的工具。
方法：将五种藻混合免疫Balb/c小鼠，加强免疫三次后，取血清效价高的小鼠脾脏细胞按一定的比例与SP2/0骨髓瘤细胞混合，加入PEG4000使两者融合。用HAT，HT等选择性培养基培养杂交瘤细胞，然后用间接血凝初步筛选出有抗体分泌的细胞克隆。进一步采用间接ELISA法等，用海洋褐胞藻等五种赤潮藻细胞包被96孔酶标板筛选分泌特异性单克隆抗体的克隆。扩增阳性克隆并冻存，制备腹水，测定腹水效价，用硫酸氨，protein-A等法纯化腹水型抗体。
结果：所得杂交瘤细胞融合率为37.5％。经反相间接血凝、间接ELISA法等筛选到能分泌抗藻单克隆抗体的细胞株，其中9AD2B2对海洋褐胞藻和卵圆褐胞藻的特异性高；15CA7对赤潮异弯藻和尖刺拟菱形藻的特异性高；23BG2和23BG9对赤潮异弯藻、具齿原甲藻与尖刺拟菱形藻的特异性高。而获得的M18号杂交瘤细胞株分泌的单克隆抗体可与以上五种藻产生高效价的明显交叉反应，显示此单抗可代替五种海洋藻的抗血清。在藻最佳包被浓度时，9AD2A8腹水对五种藻的效价均可达到1：3200，23BG9E11腹水五种藻的效价可达1：1280，两种腹水主要成分均为IgG1。
结论：利用杂交瘤技术能制备出分泌抗五种藻的单克隆抗体的细胞株。该类细胞株分泌的单克隆抗体可特异性地结合到五种藻细胞上，这意味着可用此类单克隆抗体鉴定、检测这五种赤潮藻。
Objective: To obtain several lines of cells that can secrete highly specific, affinitive monoclonal antibodies against five kinds of algal cells which are Chattonella marina, Chattonella ovata, Hetewsigma akashiwo, Prorocentrum dentatum and Pseudonitzschia pungens, respectively. The monoclonal antibodies secreted by such hybridomas can specifically bind to algal cells, which implies we can use them to determine the five kinds of algae showed above, and this technology can provide an effective tool for detecting and preventing red tide.
Methods: The 5 kinds of algae were mixed well to immunize BABL/c mice and boosted for 3 times. Splenocytes got from mice whose antisera showed high liters were fused with myleoma cells Sp2/0 in some ratio by dipping polyethylene glycol(PEG)4000. The hybridomas were cultivated in selection media HAT, HT, then screened by reverse indirect agglutination test. Furthermore, by indirect ELISA, we coated 96-well microplates with Chattonella marina, Chattonella ovata, Hetewsigma akashiwo and so on to screen clones which could secrete specific monoclonal antibodies. The positive clones were expanded and cryopreserved. The ascites were produced, and their titers were determined, then purified with ammonium sulfale and protein-A.
Results: The hybridoma cloning ratio was aboul 37.5 percent Screening by reverse indirecl agglutination lesl, indirecl ELISA, elc, we oblained several cell lines that could secrete specific monoclonal antibodies against algae, of which 9AD2B2 against Chattonella marina and Chattonella ovata, 15CA7 against Hetewsigma akashiwo and Pseudonitzschia pungens, 23BG2 and 23BG9 against Hetewsigma akashiwo, Prorocentrum dentatum and Pseudonitzschia pungens. And tthe monoclonal antibodies from clone M18 showed highly significant cross-reactivity among such five kinds of algae, which indicated that they could be used as candidates for antisera against Ihe 5 kinds of algae. In the optimum coating concentration of algae, the tiler of from 9AD2A8 was 1:3200, and the titer of 23BG9E11 was l:1280.And the main ingredients of such two ascites were IgG1(s).
Conclusion: It was feasible to obtain hybridoma cell lines which could secrete
monoclonal antibodies against the five kinds of algae by hybridoma technology. The monoclonal antibodies secreted by such hybridomas could specifically bind to the five kinds of algae, which implied that they could be used to determine and detect such five kinds of red tide algae.
方法：将五种藻混合免疫Balb/c小鼠，加强免疫三次后，取血清效价高的小鼠脾脏细胞按一定的比例与SP2/0骨髓瘤细胞混合，加入PEG4000使两者融合。用HAT，HT等选择性培养基培养杂交瘤细胞，然后用间接血凝初步筛选出有抗体分泌的细胞克隆。进一步采用间接ELISA法等，用海洋褐胞藻等五种赤潮藻细胞包被96孔酶标板筛选分泌特异性单克隆抗体的克隆。扩增阳性克隆并冻存，制备腹水，测定腹水效价，用硫酸氨，protein-A等法纯化腹水型抗体。
结果：所得杂交瘤细胞融合率为37.5％。经反相间接血凝、间接ELISA法等筛选到能分泌抗藻单克隆抗体的细胞株，其中9AD2B2对海洋褐胞藻和卵圆褐胞藻的特异性高；15CA7对赤潮异弯藻和尖刺拟菱形藻的特异性高；23BG2和23BG9对赤潮异弯藻、具齿原甲藻与尖刺拟菱形藻的特异性高。而获得的M18号杂交瘤细胞株分泌的单克隆抗体可与以上五种藻产生高效价的明显交叉反应，显示此单抗可代替五种海洋藻的抗血清。在藻最佳包被浓度时，9AD2A8腹水对五种藻的效价均可达到1：3200，23BG9E11腹水五种藻的效价可达1：1280，两种腹水主要成分均为IgG1。
结论：利用杂交瘤技术能制备出分泌抗五种藻的单克隆抗体的细胞株。该类细胞株分泌的单克隆抗体可特异性地结合到五种藻细胞上，这意味着可用此类单克隆抗体鉴定、检测这五种赤潮藻。
Objective: To obtain several lines of cells that can secrete highly specific, affinitive monoclonal antibodies against five kinds of algal cells which are Chattonella marina, Chattonella ovata, Hetewsigma akashiwo, Prorocentrum dentatum and Pseudonitzschia pungens, respectively. The monoclonal antibodies secreted by such hybridomas can specifically bind to algal cells, which implies we can use them to determine the five kinds of algae showed above, and this technology can provide an effective tool for detecting and preventing red tide.
Methods: The 5 kinds of algae were mixed well to immunize BABL/c mice and boosted for 3 times. Splenocytes got from mice whose antisera showed high liters were fused with myleoma cells Sp2/0 in some ratio by dipping polyethylene glycol(PEG)4000. The hybridomas were cultivated in selection media HAT, HT, then screened by reverse indirect agglutination test. Furthermore, by indirect ELISA, we coated 96-well microplates with Chattonella marina, Chattonella ovata, Hetewsigma akashiwo and so on to screen clones which could secrete specific monoclonal antibodies. The positive clones were expanded and cryopreserved. The ascites were produced, and their titers were determined, then purified with ammonium sulfale and protein-A.
Results: The hybridoma cloning ratio was aboul 37.5 percent Screening by reverse indirecl agglutination lesl, indirecl ELISA, elc, we oblained several cell lines that could secrete specific monoclonal antibodies against algae, of which 9AD2B2 against Chattonella marina and Chattonella ovata, 15CA7 against Hetewsigma akashiwo and Pseudonitzschia pungens, 23BG2 and 23BG9 against Hetewsigma akashiwo, Prorocentrum dentatum and Pseudonitzschia pungens. And tthe monoclonal antibodies from clone M18 showed highly significant cross-reactivity among such five kinds of algae, which indicated that they could be used as candidates for antisera against Ihe 5 kinds of algae. In the optimum coating concentration of algae, the tiler of from 9AD2A8 was 1:3200, and the titer of 23BG9E11 was l:1280.And the main ingredients of such two ascites were IgG1(s).
Conclusion: It was feasible to obtain hybridoma cell lines which could secrete
monoclonal antibodies against the five kinds of algae by hybridoma technology. The monoclonal antibodies secreted by such hybridomas could specifically bind to the five kinds of algae, which implied that they could be used to determine and detect such five kinds of red tide algae.
引文
[1] 郑重，李少菁，许振祖．海洋浮游生物学[M]．北京：海洋出版社，1984(第一版)：19-33
[2] 宋杰军，毛庆武．海洋生物毒素学[M]．北京：北京科学技术出版社，1996(第一版)：493-498
[3] Anderson D M, Sullivan J J, Reguera B. Paralytic shellfish poisoning in northwest Spain: the toxicity of the dinoflagellate Gymnodinium catenatum [J]. Toxicon, 1989, 27(6): 665-674
[4] Kodama M, Ogata M, Fukuyo Y, et al. Protogonyaulax cohorticula, a toxic dinoflagellate found in the gulf of Thailand [J]. Toxicon, 1988, 26(8): 707-712
[5] Karunasagar I K I, Qshima Y, Yasumoto T. A toxin profile for shellfish involed in an Outbreak of paralytic shellfish poisoning in India[J]. Toxicon, 1990, 28(7): 868-870
[6] Anderson D M, Kulis D M, Qi Y Z, et al. Paralytic shellfish poisoning in southern China[J]. Toxicon, 1996, 34(5): 579-590
[7] 林燕棠，杨美兰，陈瑞雯等．广东沿海麻痹性贝类毒素的研究[J]．海洋与湖沼．1994，21(2)：220-225
[8] Zhou M J, Li J, Bernd Luckas et al. A recent shellfish toxin investigation in china [J]. Marine Pollution Bulletin, 1999, 39(1-12): 331-334
[9] 周名江，朱明远，张经．中国赤潮的发生趋势和研究进展[J]．生命科学，2001，13(2)：53-59
[10] 郑重．海洋浮游生物生态学文集[M]．厦门：厦门大学出版社，1986(第一版)：4-5
[11] Chen C Y, Chou H N. Transmission of the paralytic shellfish poisoning toxins, from dinofiagellate to gastropod [J]. Toxicon, 1998, 36(3): 515-522
[12] Morono A, Fraco J, Miranda M, et al. The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels(Mytilus galloprovincialis Lmk) [J]. Journal of Experimental Marine Biology and Ecology, 2001, 257:117-132
[13] Takeshi Y. Marine toxins [J]. Chemistry Review, 1993, 5:1897
[14] 曾呈奎，相建海．海洋生物技术[M]．山东：山东科学技术出版社，1998(第一版)：416-429
[15] Soames M C P. Shellfish poisoning [J]. Chemistry Review, 1995, 62(2): 22
[16] 费鸿年．发生赤潮的原因[J]．学艺，1952，22(1)：1-3
[17] 周贞英．平潭岛的东洋水一束毛藻(Trichodesmium erythraeum Her．)[J)．福建师范学院学报，1962，(4)：75-79
[18] Qi Y, Zhang Z, Hong Y, et al. Occurrence of red tides on the coasts of China [A]. Smayda T J, Shimizu Y, (eds.)Toxic Marine Phytoplankton[C]. Amsterdam:Elsevier, 1993:284-287
[19] 邓磊，齐雨藻，骆育敏．大鹏湾有毒藻生物胞囊研究[J]．暨南大学学报(自然科学版)，1995，16(1)：121-126
[20] 陈月琴，屈良鹄，曾陇酶，齐雨藻，邓磊．南海赤潮有毒甲藻链状—塔马亚历山大藻的分子鉴定[J]．海洋学报，1999，21(3)：106-111
[21] 陈月琴，屈良鹄，邱小忠，曾陇酶，齐雨藻．甲藻单个细胞DNA的制备及在赤潮藻类分子鉴定中的应用[J]．中山大学学报(自然科学版)，1997，36(4)：66-69
[22] 何家菀，陈明慧，何振荣．小定鞭藻毒素的分离与鉴定[J]．水生生物学报，1996，20(1)：41-48
[23] 夏综万，于斌，史建辉．大鹏湾的赤潮生态仿真模型[J]．海洋与湖沼，1997，28(5)：468-474
[24] 俞志明，宋秀贤，张波，孙晓霞．粘土表面改性及对赤潮生物絮凝作用的研究[J]．科学通报，1999，44(3)：308-311
[25] Hallegraeff G M., Anderson D M., Cembella A D. Manual on Harmful Marine Microalgae [M]. Denmark:UNESCO, 1995
[26] 齐雨藻，洪英，吕颂辉，朱从举，李雅琴，梁松．中国赤潮生物新记录种——海洋褐胞藻[J]．暨南大学学报，1991，12(3)：92-85
[27] 齐雨藻，洪笑，吕颂辉等．南海大鹏湾海洋褐胞藻赤潮及其成因[J]．海洋与湖沼．1994，25(2)：32-137
[28] 齐雨藻，楚建华，黄奕华．诱发海洋褐胞藻赤潮的环境因素分析．海洋通报．1993，12(2)：30-34
[29] 矫晓阳，郭皓．中国北黄海发生的两次海洋褐胞藻赤潮[J]．海洋环境科学．1996，15(3)：41-45
[30] Marshall J A, Newman S. Differences in photoprotective pigment production between Japanese and Australian lines of Chanonella marina (Raphidophyceae) [J]. Journal of Experimental Marine Biology and Ecology. 2002, 272(1): 13-27
[31] Onoue Y, Nozawa K. Separation of toxins from harmful red tides occurring along the coast of Kagoshima Prefecture [A]. Okaichi T., D M Anderson, T nemoto(eds),Red Tides: Biology, Environmental Science and Toxicology [C]. New York, Elsevier Science Publishing Co., 1989:371-374
[32] Endo M. Y Onoue. A Kuroki. Neurotoxin-induced cardiac disorder and its role in the death of fish exposed to Chattonella marina [J]. Mar Biol. 1992, 112(3): 371-376
[33] Nakanishi K, Karube I, Hiroshi S, Uchida A. Ishida, Y. Japan - Immunosensor detection of the red tide-causing plankton [J]. Biosensors and Bioelectronics 1996, 11(11): ix-x
[34] Nakanishi K, Karube I, Hiroshi S, et al. Detection of the red tide-causing plankton Chattonella marina using a piezoelectric immunosensor [J]. Analytic Chemica Acta, 1996, 325:73-80
[35] Nagasaki K. An identification of genus Chattonella by means of monoclonal antibodies [J]. Bull Nansei Natl Fish Res Inst. 1993, (26): 133-183
[36] 王惠卿．大连市黑石礁湾赤潮调查与研究[J]．环境监测管理与技术，1994，6(4)：13-16
[37] 郭玉洁，大连赤潮生物——赤潮异弯藻[J]．海洋与湖沼．1994，25(2)：211-215
[38] 李才文，管越强，于仁诚，张波，俞志明．赤潮异弯藻对中国对虾感染白斑综合症病毒的影响[J]．海洋学报，2003，25(1)：132-137
[39] Bell, Michael V.;Dick, James R.;Pond,David W. Octadecapentaenoic acid in a raphidophyte algae, Heterosigma akashiwo [J]. Phytochemistry. 1997, 45(2): 303 -306
[40] Itakura S, Nagasaki K. Yamaguchi M, Imai I. Cyst formation in the red tide flagellate Heterosigma akashiwo (Raphidophyceae) [J]. Oceanographic Literature Review. 1997, 44(5): 480
[41] Hard J J, Connell L, Hershberger W K, Harrell L W. Genetic variation in mortality of chinook salmon during a bloom of the marine alga Heterosigma akashiwo [J]. Journal of Fish Biology. 2000, 56(6): 1387-1397
[42] Kamiyama T. Change in the microzooplankton community during decay of a Heterosigma akashiwo bloom [J]. Oceanographic Literature Review. 1996, 43(1): 57
[43] Seki Hideshi, Suzuki Akira,Iburi Yasuyuki. Biosorption of heavy metal ions to a marine microalga, Heterosigma akashiwo (Hada) Hada [J]. Journal of Colloid and Interface Science. 2000, 229(1): 196-198
[44] Shono Mariko, Wada Masato, Hara Yukichi, Fujii Tadashi. Molecular cloning of Na+-ATPase cDNA from a marine alga, Heterosigma akashiwo [J]. Biochimica et Biophysica Acta (BBA )/Biomembranes, 2001, 1511(1): 193-199
[45] Asai Ryoichi, Matsukawa Ritsuko, Ikebukuro Kazunori, Karube Isao. Highly sensitive chemiluminescence flow-injection detection of the red tide phytoplankton Heterosigma carterae [J]. Analytica Chimica Acta., 1999, 390(1-3): 237-244
[46] John V. Tyrrell, Laurie B. Connell, Chris A. Scholin. Monitoring for Heterosigma akashiwo using a sandwich hybridization assay [J]. Harmful Algae, 2002, 1:205-214
[47] Carmedo R. Tomas. Identifying Marine Phytoplankton [M]. California:Academic Press, 1997:422～431
[48] Yasuwo Fukuyo, Hideaki Takano, Mitsuo Chihara, Kazumi Matsuoka. Red tide organisms in Japan-An illustrated taxonomic guide [M]. Japan:Uchida Rokakuho, 1990, 26～27
[49] 金德祥，陈金环，黄凯歌．中国海洋浮游硅藻类[M]．上海：上海科学技术出版社，1965(第一版)：205-207
[50] Lundholm N, Skov J, Pocklington R, Moestrup φ. Domoic acid, the toxic amino acid responsible for amnesic shellfish poisoning, now in Pseudo-nitzschia seriata (Bacillariophyceae) in Europe [J]. Phycologia. 1994, 33: 475-478.
[51] Bates S S. Domoic acid and Pseudo-nitzschia references [A]. A.M. Wood and L.M. Shapiro(eds.), Domoic acid, final riport of the workshop, 1993, 13-21
[52] Bates S S, Bird C J, Frettas A S W de, Foxall R, Gilgan M, et al. Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shellfish from
eastern Prince Edward Island, Canada [J]. Can. J. Fish. Aquat. Sci. 1989, 48: 1203-1215
[53] Hasle, Grethe R. Are most of the domoic acid-producing species of the diatom genus Pseudo-nitzschia cosmopolites? [J]. Harmful algae, 2002, 1(2): 137-146
[54] Jones T O, Whyte J N C, Ginther N G, Townsend L D, Iwama G K. Haemocyte changes in the Pacific oyster Crassostrea gigas, caused by exposure to domoic acid in the diatom Pseudoni-tzschia pungens f. multiseries [J]. Toxicon, 1995, 33(3): 347-353
[55] Jones T O, Whyte J N C, Townsend L D, Ginther N G, Iwama G K. Effects of domoic acid on haemolymph pH, PCO_2 and PO_2 in the Pacific oyster, Crassostrea gigas and the California mussel, Mytilus californianus [J]. Aquatic Toxicology, 1995, 31(1): 43-55
[56] 俞志明，粘土矿物对尖刺拟菱形藻多裂型生长和藻毒素产生的影响[J]．海洋与湖沼，1998，29(1)：47-52
[57] Hasle G R. Pseudo-nitzschia as a genus distinct from Nitzschia (Bacillariophyceae) [J]. Phycol. 1994, 30:1036-1039
[58] Hasle G R. Pseudo-nitzschia pungens and P. multiseries (Bacillariophyceae): nomen --clatural history, morphology, and distribution [J]. Phycol. 1995, 31:428-435
[59] 张诚，邹景忠．尖刺拟菱形藻氮磷吸收动力学以及氮磷限制下的增殖特征[J]．海洋与湖沼，1997，28(6)：59-63
[60] Manhart J, Fryxell G A, Villac M C, Segura L Y. Pseudo-nitzschia pungens and P. multiseries (Bacillariohphyceae): nuclear ribosomal DNAs and species differences [J]. Phycol. 1995, 31:421-427
[61] Sako Y, Adachi M, Ahd, et al. Specific monoclonal antibodis and DNA probes for the identification of the toxic dinoflagellate genus Alexandrium [A]. P. Lassus, G Arzul, E. Eard, P. Gentien and C. Marcaillou, eds. Harmful Marine Algal Blooms [C]. Paris Lavoisier, 1995, 77-81
[62] HALLEGRAEFF G M. In Vitro biochemical and cellular assays [J]. Intergovernmental Oceanographic Commission Manuals and Guides. 1995, 33: 182-190
[63] Mountfort D, Suzukit T, Truman P. Protein phosphatase ihibition assay adapted for
determination of total DSP in contaminated mussels [J]. Toxicon, 2001, 39(2-3): 383-393
[64] Sako Y, Adachi M, Ishida Y. Preparation and characterization of monoclonal antibodies to Alexandrium species [A]. Smayda T J., Shimizu Y(eds), Toxic phytoplankton blooms in the sea[C]. New York, Elsevier, 1993(1st version): 87-93
[65] 徐志凯．实用单克隆抗体技术[M]．西安：陕西科学技术出版社，1992(第一版)：9-83
[66] 沈关心，周汝麟．现代免疫学实验技术[M]．武汉：湖北科学技术出版社，2002(第二版)：23-25
[67] Khler G, Milstein C. Continuous culture of fused cells secreting antibody of redefined specificity [J]. Nature. 1975, 256-495
[68] Zola H. Monoclonal Antibodies. Oxford, UK:BIOS Scientific Publishers Limited, 2000(1st version): 24-26
[69] 薛庆善．体外培养的原理与技术[M]．北京：科学出版社，2002(第一版)：24-26
[70] 程宝鸾．动物细胞培养技术[M]．广州：华南理工大学出版社，2001(第一版)：107-110
[71] 朱倍坤．免疫酶技术[M]．济南：山东科学技术出版社，1983(第一版)：89-99
[72] 将成淦．酶免疫测定法[M]．北京：人民卫生出版社，1984(第一版)：83-133
[73] Nagasaki K, Uchida A, Hiroishi S, et al. Current Topics in Marine Biotechnology [M]. Tokyo, Fuji Technology Press, 1989: 164.
[74] Córdova J L, Jamett A, Lembeye G S. Immunolocalization of theca antigens on Alexandrium catenella and their use to isolate cells from fixed phytoplankton samples [J]. Harmful Algae, 2002, 1 (4):351-360
[75] 杨景山．医学细胞化学与细胞生物技术[M]．北京：北京医科大学、中国协和医科大学联合出版社，1990(第一版)：133-145
[76] 蔡文琴，王伯沄．实用免疫细胞化学[M]．成都：四川科学技术出版社，1988(第一版)：38-76
[77] Nishitani, Goh; Sugioka, Hikaru; Imai, Ichiro. Seasonal distribution of species of the toxic dinoflagellate genus Dinophysis in Maizuru Bay (Japan), with comments on their autofluorescence and attachment of picophytoplankton [J]. Harmful Algae,
2002，1(3)：253-264
[78] Wulf B．Storch，阮幼冰．免疫荧光基础—实验新原理及其临床新应用[M]．北京：人民卫生出版社，2000(第二版)：28-38
[79] 刘令梅．赤潮的检测技术和防治措施[J]．海洋技术，1998，17(3)：58-64
[80] Godhe A, Rehnstam-Holm Ann-Sofi, Karunasagar I, Karunasagar I. PCR detection of dinoflagellate cysts in field sediment samples from tropic and temperate enviro-nments [J]. Harmful Algae, 2002, 1(4): 361-373
[81] Córdova J L, Müller I. Use of PCR and partial sequencing of the large-subunit rRNA gene to identify Alexandrium catenella(dinophyceae) from the south of Chile[J]. Harmful Algae, 2002, 1(4): 343-350
[82] Godhe, Anna; Anderson, Donald M.; Rehnstam-Holm, Ann-Sofi. PCR amplification of microalgal DNA for sequencing and species identification: studies on fixatives and algal growth stages [J]. Harmful Algae, 2002, 1(4): 375-382
[2] 宋杰军，毛庆武．海洋生物毒素学[M]．北京：北京科学技术出版社，1996(第一版)：493-498
[3] Anderson D M, Sullivan J J, Reguera B. Paralytic shellfish poisoning in northwest Spain: the toxicity of the dinoflagellate Gymnodinium catenatum [J]. Toxicon, 1989, 27(6): 665-674
[4] Kodama M, Ogata M, Fukuyo Y, et al. Protogonyaulax cohorticula, a toxic dinoflagellate found in the gulf of Thailand [J]. Toxicon, 1988, 26(8): 707-712
[5] Karunasagar I K I, Qshima Y, Yasumoto T. A toxin profile for shellfish involed in an Outbreak of paralytic shellfish poisoning in India[J]. Toxicon, 1990, 28(7): 868-870
[6] Anderson D M, Kulis D M, Qi Y Z, et al. Paralytic shellfish poisoning in southern China[J]. Toxicon, 1996, 34(5): 579-590
[7] 林燕棠，杨美兰，陈瑞雯等．广东沿海麻痹性贝类毒素的研究[J]．海洋与湖沼．1994，21(2)：220-225
[8] Zhou M J, Li J, Bernd Luckas et al. A recent shellfish toxin investigation in china [J]. Marine Pollution Bulletin, 1999, 39(1-12): 331-334
[9] 周名江，朱明远，张经．中国赤潮的发生趋势和研究进展[J]．生命科学，2001，13(2)：53-59
[10] 郑重．海洋浮游生物生态学文集[M]．厦门：厦门大学出版社，1986(第一版)：4-5
[11] Chen C Y, Chou H N. Transmission of the paralytic shellfish poisoning toxins, from dinofiagellate to gastropod [J]. Toxicon, 1998, 36(3): 515-522
[12] Morono A, Fraco J, Miranda M, et al. The effect of mussel size, temperature, seston volume, food quality and volume-specific toxin concentration on the uptake rate of PSP toxins by mussels(Mytilus galloprovincialis Lmk) [J]. Journal of Experimental Marine Biology and Ecology, 2001, 257:117-132
[13] Takeshi Y. Marine toxins [J]. Chemistry Review, 1993, 5:1897
[14] 曾呈奎，相建海．海洋生物技术[M]．山东：山东科学技术出版社，1998(第一版)：416-429
[15] Soames M C P. Shellfish poisoning [J]. Chemistry Review, 1995, 62(2): 22
[16] 费鸿年．发生赤潮的原因[J]．学艺，1952，22(1)：1-3
[17] 周贞英．平潭岛的东洋水一束毛藻(Trichodesmium erythraeum Her．)[J)．福建师范学院学报，1962，(4)：75-79
[18] Qi Y, Zhang Z, Hong Y, et al. Occurrence of red tides on the coasts of China [A]. Smayda T J, Shimizu Y, (eds.)Toxic Marine Phytoplankton[C]. Amsterdam:Elsevier, 1993:284-287
[19] 邓磊，齐雨藻，骆育敏．大鹏湾有毒藻生物胞囊研究[J]．暨南大学学报(自然科学版)，1995，16(1)：121-126
[20] 陈月琴，屈良鹄，曾陇酶，齐雨藻，邓磊．南海赤潮有毒甲藻链状—塔马亚历山大藻的分子鉴定[J]．海洋学报，1999，21(3)：106-111
[21] 陈月琴，屈良鹄，邱小忠，曾陇酶，齐雨藻．甲藻单个细胞DNA的制备及在赤潮藻类分子鉴定中的应用[J]．中山大学学报(自然科学版)，1997，36(4)：66-69
[22] 何家菀，陈明慧，何振荣．小定鞭藻毒素的分离与鉴定[J]．水生生物学报，1996，20(1)：41-48
[23] 夏综万，于斌，史建辉．大鹏湾的赤潮生态仿真模型[J]．海洋与湖沼，1997，28(5)：468-474
[24] 俞志明，宋秀贤，张波，孙晓霞．粘土表面改性及对赤潮生物絮凝作用的研究[J]．科学通报，1999，44(3)：308-311
[25] Hallegraeff G M., Anderson D M., Cembella A D. Manual on Harmful Marine Microalgae [M]. Denmark:UNESCO, 1995
[26] 齐雨藻，洪英，吕颂辉，朱从举，李雅琴，梁松．中国赤潮生物新记录种——海洋褐胞藻[J]．暨南大学学报，1991，12(3)：92-85
[27] 齐雨藻，洪笑，吕颂辉等．南海大鹏湾海洋褐胞藻赤潮及其成因[J]．海洋与湖沼．1994，25(2)：32-137
[28] 齐雨藻，楚建华，黄奕华．诱发海洋褐胞藻赤潮的环境因素分析．海洋通报．1993，12(2)：30-34
[29] 矫晓阳，郭皓．中国北黄海发生的两次海洋褐胞藻赤潮[J]．海洋环境科学．1996，15(3)：41-45
[30] Marshall J A, Newman S. Differences in photoprotective pigment production between Japanese and Australian lines of Chanonella marina (Raphidophyceae) [J]. Journal of Experimental Marine Biology and Ecology. 2002, 272(1): 13-27
[31] Onoue Y, Nozawa K. Separation of toxins from harmful red tides occurring along the coast of Kagoshima Prefecture [A]. Okaichi T., D M Anderson, T nemoto(eds),Red Tides: Biology, Environmental Science and Toxicology [C]. New York, Elsevier Science Publishing Co., 1989:371-374
[32] Endo M. Y Onoue. A Kuroki. Neurotoxin-induced cardiac disorder and its role in the death of fish exposed to Chattonella marina [J]. Mar Biol. 1992, 112(3): 371-376
[33] Nakanishi K, Karube I, Hiroshi S, Uchida A. Ishida, Y. Japan - Immunosensor detection of the red tide-causing plankton [J]. Biosensors and Bioelectronics 1996, 11(11): ix-x
[34] Nakanishi K, Karube I, Hiroshi S, et al. Detection of the red tide-causing plankton Chattonella marina using a piezoelectric immunosensor [J]. Analytic Chemica Acta, 1996, 325:73-80
[35] Nagasaki K. An identification of genus Chattonella by means of monoclonal antibodies [J]. Bull Nansei Natl Fish Res Inst. 1993, (26): 133-183
[36] 王惠卿．大连市黑石礁湾赤潮调查与研究[J]．环境监测管理与技术，1994，6(4)：13-16
[37] 郭玉洁，大连赤潮生物——赤潮异弯藻[J]．海洋与湖沼．1994，25(2)：211-215
[38] 李才文，管越强，于仁诚，张波，俞志明．赤潮异弯藻对中国对虾感染白斑综合症病毒的影响[J]．海洋学报，2003，25(1)：132-137
[39] Bell, Michael V.;Dick, James R.;Pond,David W. Octadecapentaenoic acid in a raphidophyte algae, Heterosigma akashiwo [J]. Phytochemistry. 1997, 45(2): 303 -306
[40] Itakura S, Nagasaki K. Yamaguchi M, Imai I. Cyst formation in the red tide flagellate Heterosigma akashiwo (Raphidophyceae) [J]. Oceanographic Literature Review. 1997, 44(5): 480
[41] Hard J J, Connell L, Hershberger W K, Harrell L W. Genetic variation in mortality of chinook salmon during a bloom of the marine alga Heterosigma akashiwo [J]. Journal of Fish Biology. 2000, 56(6): 1387-1397
[42] Kamiyama T. Change in the microzooplankton community during decay of a Heterosigma akashiwo bloom [J]. Oceanographic Literature Review. 1996, 43(1): 57
[43] Seki Hideshi, Suzuki Akira,Iburi Yasuyuki. Biosorption of heavy metal ions to a marine microalga, Heterosigma akashiwo (Hada) Hada [J]. Journal of Colloid and Interface Science. 2000, 229(1): 196-198
[44] Shono Mariko, Wada Masato, Hara Yukichi, Fujii Tadashi. Molecular cloning of Na+-ATPase cDNA from a marine alga, Heterosigma akashiwo [J]. Biochimica et Biophysica Acta (BBA )/Biomembranes, 2001, 1511(1): 193-199
[45] Asai Ryoichi, Matsukawa Ritsuko, Ikebukuro Kazunori, Karube Isao. Highly sensitive chemiluminescence flow-injection detection of the red tide phytoplankton Heterosigma carterae [J]. Analytica Chimica Acta., 1999, 390(1-3): 237-244
[46] John V. Tyrrell, Laurie B. Connell, Chris A. Scholin. Monitoring for Heterosigma akashiwo using a sandwich hybridization assay [J]. Harmful Algae, 2002, 1:205-214
[47] Carmedo R. Tomas. Identifying Marine Phytoplankton [M]. California:Academic Press, 1997:422～431
[48] Yasuwo Fukuyo, Hideaki Takano, Mitsuo Chihara, Kazumi Matsuoka. Red tide organisms in Japan-An illustrated taxonomic guide [M]. Japan:Uchida Rokakuho, 1990, 26～27
[49] 金德祥，陈金环，黄凯歌．中国海洋浮游硅藻类[M]．上海：上海科学技术出版社，1965(第一版)：205-207
[50] Lundholm N, Skov J, Pocklington R, Moestrup φ. Domoic acid, the toxic amino acid responsible for amnesic shellfish poisoning, now in Pseudo-nitzschia seriata (Bacillariophyceae) in Europe [J]. Phycologia. 1994, 33: 475-478.
[51] Bates S S. Domoic acid and Pseudo-nitzschia references [A]. A.M. Wood and L.M. Shapiro(eds.), Domoic acid, final riport of the workshop, 1993, 13-21
[52] Bates S S, Bird C J, Frettas A S W de, Foxall R, Gilgan M, et al. Pennate diatom Nitzschia pungens as the primary source of domoic acid, a toxin in shellfish from
eastern Prince Edward Island, Canada [J]. Can. J. Fish. Aquat. Sci. 1989, 48: 1203-1215
[53] Hasle, Grethe R. Are most of the domoic acid-producing species of the diatom genus Pseudo-nitzschia cosmopolites? [J]. Harmful algae, 2002, 1(2): 137-146
[54] Jones T O, Whyte J N C, Ginther N G, Townsend L D, Iwama G K. Haemocyte changes in the Pacific oyster Crassostrea gigas, caused by exposure to domoic acid in the diatom Pseudoni-tzschia pungens f. multiseries [J]. Toxicon, 1995, 33(3): 347-353
[55] Jones T O, Whyte J N C, Townsend L D, Ginther N G, Iwama G K. Effects of domoic acid on haemolymph pH, PCO_2 and PO_2 in the Pacific oyster, Crassostrea gigas and the California mussel, Mytilus californianus [J]. Aquatic Toxicology, 1995, 31(1): 43-55
[56] 俞志明，粘土矿物对尖刺拟菱形藻多裂型生长和藻毒素产生的影响[J]．海洋与湖沼，1998，29(1)：47-52
[57] Hasle G R. Pseudo-nitzschia as a genus distinct from Nitzschia (Bacillariophyceae) [J]. Phycol. 1994, 30:1036-1039
[58] Hasle G R. Pseudo-nitzschia pungens and P. multiseries (Bacillariophyceae): nomen --clatural history, morphology, and distribution [J]. Phycol. 1995, 31:428-435
[59] 张诚，邹景忠．尖刺拟菱形藻氮磷吸收动力学以及氮磷限制下的增殖特征[J]．海洋与湖沼，1997，28(6)：59-63
[60] Manhart J, Fryxell G A, Villac M C, Segura L Y. Pseudo-nitzschia pungens and P. multiseries (Bacillariohphyceae): nuclear ribosomal DNAs and species differences [J]. Phycol. 1995, 31:421-427
[61] Sako Y, Adachi M, Ahd, et al. Specific monoclonal antibodis and DNA probes for the identification of the toxic dinoflagellate genus Alexandrium [A]. P. Lassus, G Arzul, E. Eard, P. Gentien and C. Marcaillou, eds. Harmful Marine Algal Blooms [C]. Paris Lavoisier, 1995, 77-81
[62] HALLEGRAEFF G M. In Vitro biochemical and cellular assays [J]. Intergovernmental Oceanographic Commission Manuals and Guides. 1995, 33: 182-190
[63] Mountfort D, Suzukit T, Truman P. Protein phosphatase ihibition assay adapted for
determination of total DSP in contaminated mussels [J]. Toxicon, 2001, 39(2-3): 383-393
[64] Sako Y, Adachi M, Ishida Y. Preparation and characterization of monoclonal antibodies to Alexandrium species [A]. Smayda T J., Shimizu Y(eds), Toxic phytoplankton blooms in the sea[C]. New York, Elsevier, 1993(1st version): 87-93
[65] 徐志凯．实用单克隆抗体技术[M]．西安：陕西科学技术出版社，1992(第一版)：9-83
[66] 沈关心，周汝麟．现代免疫学实验技术[M]．武汉：湖北科学技术出版社，2002(第二版)：23-25
[67] Khler G, Milstein C. Continuous culture of fused cells secreting antibody of redefined specificity [J]. Nature. 1975, 256-495
[68] Zola H. Monoclonal Antibodies. Oxford, UK:BIOS Scientific Publishers Limited, 2000(1st version): 24-26
[69] 薛庆善．体外培养的原理与技术[M]．北京：科学出版社，2002(第一版)：24-26
[70] 程宝鸾．动物细胞培养技术[M]．广州：华南理工大学出版社，2001(第一版)：107-110
[71] 朱倍坤．免疫酶技术[M]．济南：山东科学技术出版社，1983(第一版)：89-99
[72] 将成淦．酶免疫测定法[M]．北京：人民卫生出版社，1984(第一版)：83-133
[73] Nagasaki K, Uchida A, Hiroishi S, et al. Current Topics in Marine Biotechnology [M]. Tokyo, Fuji Technology Press, 1989: 164.
[74] Córdova J L, Jamett A, Lembeye G S. Immunolocalization of theca antigens on Alexandrium catenella and their use to isolate cells from fixed phytoplankton samples [J]. Harmful Algae, 2002, 1 (4):351-360
[75] 杨景山．医学细胞化学与细胞生物技术[M]．北京：北京医科大学、中国协和医科大学联合出版社，1990(第一版)：133-145
[76] 蔡文琴，王伯沄．实用免疫细胞化学[M]．成都：四川科学技术出版社，1988(第一版)：38-76
[77] Nishitani, Goh; Sugioka, Hikaru; Imai, Ichiro. Seasonal distribution of species of the toxic dinoflagellate genus Dinophysis in Maizuru Bay (Japan), with comments on their autofluorescence and attachment of picophytoplankton [J]. Harmful Algae,
2002，1(3)：253-264
[78] Wulf B．Storch，阮幼冰．免疫荧光基础—实验新原理及其临床新应用[M]．北京：人民卫生出版社，2000(第二版)：28-38
[79] 刘令梅．赤潮的检测技术和防治措施[J]．海洋技术，1998，17(3)：58-64
[80] Godhe A, Rehnstam-Holm Ann-Sofi, Karunasagar I, Karunasagar I. PCR detection of dinoflagellate cysts in field sediment samples from tropic and temperate enviro-nments [J]. Harmful Algae, 2002, 1(4): 361-373
[81] Córdova J L, Müller I. Use of PCR and partial sequencing of the large-subunit rRNA gene to identify Alexandrium catenella(dinophyceae) from the south of Chile[J]. Harmful Algae, 2002, 1(4): 343-350
[82] Godhe, Anna; Anderson, Donald M.; Rehnstam-Holm, Ann-Sofi. PCR amplification of microalgal DNA for sequencing and species identification: studies on fixatives and algal growth stages [J]. Harmful Algae, 2002, 1(4): 375-382