河豚毒素免疫学检测方法的建立及三种海洋生物毒素融合单链抗体的初步研究
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摘要
河豚毒素(Tetrodotoxin,TTX)是一种常见且危害较大的海洋生物毒素,广泛存在于海洋生物和陆栖等众多生物中,主要见于豚鱼类。豚鱼肉味鲜美,营养丰富,我国和日本等亚洲国家均有食用习惯,中毒事件频频发生,其快速检测方法的研究对预防河豚毒素中毒具有重要意义。本研究成功制备了TTX毒素人工完全抗原TTX-BSA、TTX-OVA;建立了分泌抗TTX毒素单克隆抗体杂交瘤细胞株3D4和4C5,制备并纯化了单克隆抗体(TTX-McAb),对抗体效价、亚类、分子量、亲和力、敏感性、特异性等进行了测定;建立了以单克隆抗体为基础的间接竞争ELISA、胶体金免疫层析试纸条免疫学检测方法,并组装了ELISA检测试剂盒,其性能指标能满足快速、特异、敏感的食品检测要求。为进一步探索利用融合单链抗体的海洋生物毒素高通量筛检方法,对TTX和另外2种重要的海洋贝毒素石房蛤毒素(Saxitoxin,STX)和大田软海绵酸(Okadaic acid,OA)融合单链抗体进行了研究。利用已构建的杂交瘤细胞株,RT-PCR方法获得TTX-McAb的VH和VL基因,SOE-PCR方法组装单链抗体基因TTX-ScFv(VH-Linker-VL),构建其原核表达载体TTX-ScFv-22b和真核表达载体TTX-ScFv-PICZα,获得了相应的具有反应活性的表达蛋白;将3种海洋生物毒素TTX、OA、STX单链抗体基因进行串联,构建了融合基因表达载体OST-ScFv-22b,获得了相应抗3种毒素的融合单链抗体蛋白,经ELISA检测,与3种毒素反应活性较弱。如何提高融合单链抗体反应活性使其能够用于3种毒素的同步筛检方法,有待于进一步研究。
Because okadaic acid (OA), saxitoxin (STX) and tetrodotoxin (TTX) are highly harmful to people, they are concernful marine toxins to cause outstanding safty problems of food and environment. It is important to research rapid detection methods of these toxins. In this study, the immunological methods of detecting TTX including a indirect competitive enzyme-linke immunosorbent assay (icELISA) and colloidal gold immune test paper were established, and then, fusion single chain antibody which was used to decteting OA, STX and TTX was developed. This would open up a new way for detecting marine toxins and other food pollutants .
Man-made complete antigens (TTX-OVA and TTX-BSA) were successfully prepared by methanal couple, and the conjugation ratios of TTX to carrier proteins BSA or OVA respectively were 14:1 and 6:1. BALB/c mice immuned by complete antigen(TTX-OVA)made immune response to produce antibodies against TTX. The hybridoma cells (3D4 and 4C5) which secreted monoclonal antibody (McAb) against TTX were established by monoclonal antibody technique. The characteristics of TTX-McAb were determined, such as titer, subtype, molecular weight, affinity, sensitivity and specificity. Two hybridoma cell lines were identified that secretes IgG1 monoclonal antibody against TTX, designated 3D4 and 4C5 after subcloned for 3 cycles by indirect ELISA. The average number of the hybridoma chromosomes were 50±2 and 50±4 and exceeded parent cells respectively. ELISA result showed that the titer of the cell culture supernatant were 1:640 and 1:320, antibody titer 5.12×105 and 3.2×105, and the affinity constant 6.2×106 L/mol and 5.9×106 L/mol respectively.
The TTX-McAb of 3D4 was manufactured by means of intraperitoneally injecting BALB/c mice and purified by CA-AS method. The result showed that purification ratio was up to 61.24 %, the recovery was 23.48%, and the purity of IgG was up to 90.99%. Experiments of specificity detection to STX, OA, MC and NOD by indirect ELISA was carried out, showing that TTX-McAb performed no cross reaction with OA, MC and NOD except STX. Preparation of TTX-McAb had successfully established to facilitate immunological detection and preparation of single-chain antibody.
An icELISA based on TTX-McAb was set up, detecting condition were optimized, TTX standard and mock samples were detected. Optimized ELISA reaction condition as follow: antigen covering concentration was 1.0μg/mL, antibody dilute strength 1:40 000,covering condition to stay overnight at 4℃, competition action at 37 OC for 30 min, action time of antibody enzyme marked 30 min, action time of substrate 10 min. Equation of linear regression was y=25.681x-4.2725, and coefficient correlation R2=0.989, concentration limit 0.05 ng/mL, linear range 0.05～50 ng/mL. TTX standard and mock samples of 216 portions, including crab, shellfish as well as spiral vagina muscle and egg tissue, were detected. Results showed that average recovery were respectively TTX standard samples 88.89%, crab spawn 87.05%, crab muscle 80.96%, snail 83.26%, shellfish 85.59%. Inner and interassay coefficient of variation were smaller than 7% and 10% respectively. ELISA kit of within plate was packaged, and its performance index reached to the fast,specific and sensitive detection requirement. Specificity detection to STX, OA, MC and NOD by indirect ELISA was done, and results indicated that the monoclonal antibody had no cross reaction with OA, MC and NOD except STX.
Colloidal gold immune test paper based on monoclonal antibody was developed, and TTX standard samples were detected. Colloidal gold particles of 20 nm were prepared using trisodium citrate as reducer, and colloidal gold probe was prepared, and condition of paper test paper slip was optimized. Slip of test paper was packaged. Results showed that the best coating density for antibody and colloid gold was 8.0μg/mL, the best pH value of coating the colloid gold was 8.5, the lowest detection concentration limit 50 ng/mL. Specificity detection with STX, OA, MC, NOD by indirect ELISA was done, and result indicated that the monoclonal antibody had no cross reaction with OA, Mc and NOD other than STX. The time for guarantee the quality was 8 weeks at 4℃.
VH and VL genes of TTX-McAb were amplified from the RNA of hybridoma cell by RT-PCR using VL and VH genes primers designed according to the monoclonal antibody of mouse source, and were ligated with linkers. The single chain antibody gene TTX-ScFv (VH -Linker-VL) was constructed by SOE-PCR. TTX-ScFv gene was 744 bp including VH of 366 bp, VL of 333 bp and linker of 45 bp. Result by NCBI blast analysis showed that VLand VH genes not only consisted with variable region characteristic of the monoclonal antibody of mouse, and but also had high homology with many single-chain antibody registered.
Prokaryotic expression vector TTX-ScFv-22b and eukaryotic expression vector TTX-ScFv-PICZαwere constructed and expressed proteins with reactive activity with TTX were obtained. Recombinant plasmid TTX-ScFv-22b was transferred into E.coli BL21(DE3)and gained high performance expression. Molecular wieght of expressed protein was 26.045 ku. Fusion proteins were induced to express by IPTG at 37℃for 6 h with amounting to 51.38% in inclusion, but amounting to 42.14% at 25℃for 12 h in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing to and ultrafiltrating. Activity was analysed by indirect competition ELISA(ic ELISA).
TTX-ScFv gene was cloned into expression vector pPICZαA, and recombinant expression vector TTX-ScFv-PICZαwas constructed. After linearrization, recombinant plasmid was transferred into GS115 of Pichia pastoris, and then were induced to express by methanol at 30℃for 72h with amounting to 15.10%. Expressed fusion protein were concentrated and purified by dialyzing and ultrafiltration, and determined by SDS-PAGE. Activity was analysed by icELISA. ELISA results indicated that expressed fusion proteins in prokaryotic and eukaryotic systems performed reactivity with TTX antigens, and activity of the eukaryotic expressed protein was a little higher than the prokaryotic, but both of them were lower significantly than TTX-McAb.
At the same time, three ScFv genes, including OA-ScFv, STX-ScFv and TTX-ScFv, were linked to OA-STX-TTX-ScFv by SOE-PCR. OA-STX-TTX-ScFv gene was 2 256 bp, encoding 752 amino acids. Fused single-chain antibody against three toxins were obtained. Molecular weight of expressed protein was about 79.000 ku and consisted with predicted value. Expressed fusion protein induced by IPTG at 37℃for 6 h amounted to 12.70% in inclusion, but expressed fusion protein induced by IPTG at 25℃for 12 h amounted to 10.90% in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing and ultrafiltrating. ELISA results indicated that the expressed fusion protein had weak reactive activity with three toxins respectively. Fusion single-chain antibodies were expressed with bioactivity. How to improve the bioactivity of fusion single-chain antibodies to estiblish broad-spectrum detection methods need to study in the future.
Because okadaic acid (OA), saxitoxin (STX) and tetrodotoxin (TTX) are highly harmful to people, they are concernful marine toxins to cause outstanding safty problems of food and environment. It is important to research rapid detection methods of these toxins. In this study, the immunological methods of detecting TTX including a indirect competitive enzyme-linke immunosorbent assay (icELISA) and colloidal gold immune test paper were established, and then, fusion single chain antibody which was used to decteting OA, STX and TTX was developed. This would open up a new way for detecting marine toxins and other food pollutants .
Man-made complete antigens (TTX-OVA and TTX-BSA) were successfully prepared by methanal couple, and the conjugation ratios of TTX to carrier proteins BSA or OVA respectively were 14:1 and 6:1. BALB/c mice immuned by complete antigen(TTX-OVA)made immune response to produce antibodies against TTX. The hybridoma cells (3D4 and 4C5) which secreted monoclonal antibody (McAb) against TTX were established by monoclonal antibody technique. The characteristics of TTX-McAb were determined, such as titer, subtype, molecular weight, affinity, sensitivity and specificity. Two hybridoma cell lines were identified that secretes IgG1 monoclonal antibody against TTX, designated 3D4 and 4C5 after subcloned for 3 cycles by indirect ELISA. The average number of the hybridoma chromosomes were 50±2 and 50±4 and exceeded parent cells respectively. ELISA result showed that the titer of the cell culture supernatant were 1:640 and 1:320, antibody titer 5.12×105 and 3.2×105, and the affinity constant 6.2×106 L/mol and 5.9×106 L/mol respectively.
The TTX-McAb of 3D4 was manufactured by means of intraperitoneally injecting BALB/c mice and purified by CA-AS method. The result showed that purification ratio was up to 61.24 %, the recovery was 23.48%, and the purity of IgG was up to 90.99%. Experiments of specificity detection to STX, OA, MC and NOD by indirect ELISA was carried out, showing that TTX-McAb performed no cross reaction with OA, MC and NOD except STX. Preparation of TTX-McAb had successfully established to facilitate immunological detection and preparation of single-chain antibody.
An icELISA based on TTX-McAb was set up, detecting condition were optimized, TTX standard and mock samples were detected. Optimized ELISA reaction condition as follow: antigen covering concentration was 1.0μg/mL, antibody dilute strength 1:40 000,covering condition to stay overnight at 4℃, competition action at 37 OC for 30 min, action time of antibody enzyme marked 30 min, action time of substrate 10 min. Equation of linear regression was y=25.681x-4.2725, and coefficient correlation R2=0.989, concentration limit 0.05 ng/mL, linear range 0.05～50 ng/mL. TTX standard and mock samples of 216 portions, including crab, shellfish as well as spiral vagina muscle and egg tissue, were detected. Results showed that average recovery were respectively TTX standard samples 88.89%, crab spawn 87.05%, crab muscle 80.96%, snail 83.26%, shellfish 85.59%. Inner and interassay coefficient of variation were smaller than 7% and 10% respectively. ELISA kit of within plate was packaged, and its performance index reached to the fast,specific and sensitive detection requirement. Specificity detection to STX, OA, MC and NOD by indirect ELISA was done, and results indicated that the monoclonal antibody had no cross reaction with OA, MC and NOD except STX.
Colloidal gold immune test paper based on monoclonal antibody was developed, and TTX standard samples were detected. Colloidal gold particles of 20 nm were prepared using trisodium citrate as reducer, and colloidal gold probe was prepared, and condition of paper test paper slip was optimized. Slip of test paper was packaged. Results showed that the best coating density for antibody and colloid gold was 8.0μg/mL, the best pH value of coating the colloid gold was 8.5, the lowest detection concentration limit 50 ng/mL. Specificity detection with STX, OA, MC, NOD by indirect ELISA was done, and result indicated that the monoclonal antibody had no cross reaction with OA, Mc and NOD other than STX. The time for guarantee the quality was 8 weeks at 4℃.
VH and VL genes of TTX-McAb were amplified from the RNA of hybridoma cell by RT-PCR using VL and VH genes primers designed according to the monoclonal antibody of mouse source, and were ligated with linkers. The single chain antibody gene TTX-ScFv (VH -Linker-VL) was constructed by SOE-PCR. TTX-ScFv gene was 744 bp including VH of 366 bp, VL of 333 bp and linker of 45 bp. Result by NCBI blast analysis showed that VLand VH genes not only consisted with variable region characteristic of the monoclonal antibody of mouse, and but also had high homology with many single-chain antibody registered.
Prokaryotic expression vector TTX-ScFv-22b and eukaryotic expression vector TTX-ScFv-PICZαwere constructed and expressed proteins with reactive activity with TTX were obtained. Recombinant plasmid TTX-ScFv-22b was transferred into E.coli BL21(DE3)and gained high performance expression. Molecular wieght of expressed protein was 26.045 ku. Fusion proteins were induced to express by IPTG at 37℃for 6 h with amounting to 51.38% in inclusion, but amounting to 42.14% at 25℃for 12 h in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing to and ultrafiltrating. Activity was analysed by indirect competition ELISA(ic ELISA).
TTX-ScFv gene was cloned into expression vector pPICZαA, and recombinant expression vector TTX-ScFv-PICZαwas constructed. After linearrization, recombinant plasmid was transferred into GS115 of Pichia pastoris, and then were induced to express by methanol at 30℃for 72h with amounting to 15.10%. Expressed fusion protein were concentrated and purified by dialyzing and ultrafiltration, and determined by SDS-PAGE. Activity was analysed by icELISA. ELISA results indicated that expressed fusion proteins in prokaryotic and eukaryotic systems performed reactivity with TTX antigens, and activity of the eukaryotic expressed protein was a little higher than the prokaryotic, but both of them were lower significantly than TTX-McAb.
At the same time, three ScFv genes, including OA-ScFv, STX-ScFv and TTX-ScFv, were linked to OA-STX-TTX-ScFv by SOE-PCR. OA-STX-TTX-ScFv gene was 2 256 bp, encoding 752 amino acids. Fused single-chain antibody against three toxins were obtained. Molecular weight of expressed protein was about 79.000 ku and consisted with predicted value. Expressed fusion protein induced by IPTG at 37℃for 6 h amounted to 12.70% in inclusion, but expressed fusion protein induced by IPTG at 25℃for 12 h amounted to 10.90% in inclusion and solubile. Two types of expressed proteins were concentrated and purified by urea dissolving, gradient dialyzing and ultrafiltrating. ELISA results indicated that the expressed fusion protein had weak reactive activity with three toxins respectively. Fusion single-chain antibodies were expressed with bioactivity. How to improve the bioactivity of fusion single-chain antibodies to estiblish broad-spectrum detection methods need to study in the future.
引文
[1] Miyazawa, K Noguchi. Distribution and origin of tetrodotoxin Journal of Toxicology-Toxin[J]. Reviews,2001,20: 11-33.
[2] Noguchi T, Arakawa O. Tetrodotoxin-distribution and acumulation in aquatic organisms, and cases of human intoxication[J]. Mar Drugs,2008, 6(2):220-242.
[3]郑思光,黄朝宗,卢中秋.河豚毒素中毒33例临床分析[J].浙江中西医结合杂志, 2006,16(5):300～301.
[4]马群飞,林升清,王明.烤角片干河豚毒素中毒事件的追踪调查[J].中国卫生监督杂志, 2001,8(4):145～147.
[5]郭柏坤,宫庆礼.河豚毒素的免疫检测进展[J].时珍国医国药,2006,17(4):628～629.
[6]王冠男,谢丽萍,张荣庆.河豚毒素的分布及动物对它的拮抗作用[J].海洋通报, 2007, 26(1):107～113.
[7] Yasumoto T, Nagai H, Yasumura D, et al. Interspecies distribution and possible origin of tetrodotoxin[J]. Ann N Y Acad Sci,1986,479:44-51.
[8]李世平,赵清良,赵强.野生和人工养殖暗纹东方魨不同组织中河豚毒素(TTX)含量的初步研究[J].南京师大学报(自然科学版),1998,21(3):90～94.
[9] Diener M, Christian B, Ahmed MS,et al. Determination of tetrodotoxin and its analogs in the puffer fish Takifugu oblongus from Bangladesh by hydrophilic interaction chromatography and mass-spectrometric detection[J]. Anal Bioanal Chem,2007, 389(6):1997-2002.
[10]李爱峰.液-质联用技术分析海洋生物毒素的研究[D].中国科学院海洋研究所,2005.
[11]吴韶菊,崔建洲,宫庆礼.河豚毒素的微生物起源[J],海洋科学,2005, 29 (10): 81～85.
[12]周玉,王哲.作用于钠通道受体的海产源神经毒素研究概况[J].动物医学进展,2005,26(5):36～40.
[13] Kodama M, Sato S, Ogata T, et al. Tetrodotoxin secreting glands in the skin of puffer fishes[J].Toxicon,1986,24(8):819-829.
[14]张旭东.河豚鱼含毒状祝究进展[J].中国食品卫生杂志,1997,9(1):32～37.
[15] Cheng CA, Hwang DF, Tsai YH, et al. Microflora and tetrodotoxin-producing bacteria in a gastropod, Niotha clathrata[J]. Food Chem Toxicol,1995,33(11):929-934.
[16]陈常英,丁晓琴,潘心富,等.河豚毒素(TTX)及其衍生物的电子结构和构效关系及与石房蛤毒素(STX)的比较研究[J].化学学报,1998,56:109～116.
[17] Kodama M, Noguchi T, Maruyama J, et al. Release of tetrodotoxin and paralytic shellfish poison from puffer liver by RNase[J]. J Biochem,1983,93(1):243-247.
[18] Xu QH, Zhao XN, Wei CH, Rong KT. Immunologic protection of anti-tetrodotoxinvaccines against lethal activities of oral tetrodotoxin challenge in mice[J]. Int Immunopharmacol, 2005,5(7-8):1213-1224.
[19] A Alcaraz,R e Whipple, H R Greeg, B.D. Andresen, et al. Analysis of tetrodotoxin[J]. Forensic Science International, 1999,99(1):35-45.
[20]沈晓书,顾明松,谢剑炜.河豚毒素分析检测方法研究进展[J].军事医学科学院院刊, 2006,30(3):295～298.
[21]桂英爱,王洪军,郝佳.河豚毒素及其代谢产物的研究进展[J].大连水产学院学学报, 2007,22(2):137～141.
[22]代秀梅,庾莉菊,张启明,等.河豚毒素的医药开发前景[J].药品评价, 2008(5) : 230～232.
[23]王小逸,付宏征,林文翰.河豚的化学成分研究[J].中国海洋药物,1999,69(2):4～8.
[24]赵永明,苏晓会,史清文,等.河豚毒素及其类似物的研究概况[J].河北医科大学学报,2007,28(6):474～480.
[25]张凤云,库宝善,姚海燕.河豚毒素单用及与Indoxacarb联合应用的镇痛抗炎作用[J].中国临床康复,2006,10(34):115～118.
[26]符凤英,库宝善,马行,等.河豚毒素与阿司匹林的协同镇痛作用[J].中国临床康复,2006,10(14):100～101.
[27] Daniel M,Schwaetz Z.Experimental study of tetrodotoxin:A long-activating topical anesthetic[J].A m J Ophthalmol,1998,125(4): 481- 487.
[28]杨朝坤,杨双强,谭松涛.河豚毒素停搏液减轻大鼠心肌细胞内钙超载作用的实验研究[J].第三军医大学学报,2005,27(3):192～199.
[29]陈素青,任雷鸣,黄致强.河豚毒素对大鼠和小鼠纳洛酮催促吗啡戒断症状的影响[J].中国药理学与毒理学杂志,2001,15(6):434～440.
[30]刘燕婷,雷红涛,钟青萍.河豚毒素的研究进展[J].食品研究与开发,2008,29(2):156～160.
[31]崔建洲,宫庆礼,顾谦群,等.一种高效制备河豚毒素(TTX)方法的研究[J].高技术通讯, 2005,15(4):89～94.
[32]阚丽丽,郭斌.正交试验法优选超声提取河豚毒素的研究[J].中华中医药学刊,2008,26(6):1274～1275.
[33]梁文京.一起集体河豚毒素食物中毒抢救体会[J].河北医学,2007,13(6):715～717.
[34] Kishi Y,Aratani M,Fukuyama T,et al.Synthetic studies on tetrodotoxin and related compounds.3.A stereospecific synthesis of an equivalent of acetylated tetrodamine[J]. J Am Chem Soc,1972,94(26):9217-9219.
[35] Ohyabu N, Nishikawa T, Isobe M. First asymmetric total synthesis of tetrodotoxin[J]. J Am Chem Soc,2003,125(29):8798-8805.
[36] Hinman A, Du Bois J.A stereoselective synthesis of (-)-tetrodotoxin [J]. J Am Chem Soc,2003,125(38):11510-11511.
[37] Nishikawa T,Asai M,Ohyabu N,et al.Stereocontrolled Synthesis of (-)-5,11- dideoxytetrodotoxin[J].Angewandte Chemie International Edition,1999,38(20):3081-3084.
[38] Sato K, Akai S, Sugita N,et al. Novel and stereocontrolled synthesis of (+/-)-tetrodotoxin from myo-inositol[J]. J Org Chem,2005,70(19):7496-7504.
[39] Urabe D, Nishikawa T, Isobe M. An efficient total synthesis of optically active tetrodotoxin from levoglucosenone[J].Chem Asian J,2006, 7,1(1-2):125-135.
[40] Hwang DF ,Noguchi T.Tetrodotoxin poisoning[J]. Adv Food Nutr Res,2007,52:141-236.
[41] Hamasaki K, Kogure K, Ohwada K. A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culture bioassay in combination with a water-soluble tetrazolium salt[J]. Toxicon.,1996,34(4):490-495.
[42] Davio SR,Fontelo PA.A competitive displacement assay to detect saxitoxin and tetrodotoxin [J]. Anal Biochem,1984,141(1):199-204.
[43] Strong PN,Smith JT, Keana JF. A convenient bioassay for detecting nanomolar concentrations of tetrodotoxin [J]. Toxicon,1973,11(5):433-438.
[44] Cheun B,Endo H,Hayashi T,et al. Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin[J].Biosensor Bioelectron,1996,11 (12):1185-1191.
[45] Nu?ez MT, Fischer S, Jaimovich E.A fluorimetric method to determine tetrodotoxin[J]. Anal Biochem,1976,72:320-325.
[46] Shen XS, Guo L, Gu MS,et al. Rapid determination of tetrodotoxin using microwave assisted alkaline hydrolysis and spectrofluorometric analysis[J].Spectroscopy and spectroscopy analysis,2006;26(12):2264-2267.
[47] Ho B,Yeo DS,Ding JL.A tetrodotoxin neutralizing system in the haemolymph of the horseshoe crab, Carcinoscorpius rotundicauda[J]. Toxicon,1994,32(7):755-762.
[48] Nakamura M,Yasumoto T.Tetrodotoxin derivatives in puffer fish[J].Toxicon,1985, 23(2):271-276.
[49] Ikebuchi J,Suenaga K,Kotoku S,et al. Thin-layer chromatography with flame ionization detection for the determination of tetrodotoxin in biological fluids[J]. J Chromatogr,1988,432:401-406.
[50] Nagashima Y,Nishio S,Noguchi T,et al.Detection of tetrodotoxin by thin-layer chromatography fast atom bombardment mass spectrometry[J].Anal Biochem, 1988,175 (1):258-262.
[51] Kawatsu K,Shibata T,Hamano Y.Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients[J]. Toxicon,1999,37(2):325-333.
[52] O'Leary MA, Schneider JJ, Isbister GK. Use of high performance liquid chromatography to measure tetrodotoxin in serum and urine of poisoned patients[J]. Toxicon, 2004,44(5):549-553.
[53] Shoji Y,Yotsu-Yamashita M,Miyazawa T,et al. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs:liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry[J].Anal Biochem,2001, 290(1):10-17.
[54]郑雍怡,王彦,张计,等.反相离子对高效液相色谱法测定SD大鼠血浆中河豚毒素的含量[J].分析化学,2008,36(5):588～592.
[55] Hayashida M,Hayakawa H,Wada K,et al.Sensitive determination of tetrodotoxin using column-switching liquid chromatography-mass spectrometry with electrospray ionization in mouse serum [J]. Journal of analytical Toxicology,2004,28(1):46-49.
[56] Pires OR Jr, Sebben A, Schwartz EF,et al.Occurrence of tetrodotoxin and its analogues in the Brazilian frog Brachycephalusephippium(Anura:Brachycephalidae)[J].Toxicon, 2002,40(6):761-766.
[57] Raybould TJ,Bignami GS,Inouye LK, et al.A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples[J].J Clin Lab Anal,1992,6(2):65-72.
[58] Kawatsu K,Shibata T,Hamano Y.Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients[J]. Toxicon,1999, 37(2):325-333.
[59] Neagu D,Micheli L,Palleschi G.Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination[J].Anal Bioanal Chem,2006, 385(6):1068-1074.
[60]宫慧芝,杨军.河豚毒素单抗ELISA检测试剂盒的研制[J].中国公共卫生,2005,21(12):1623～1624.
[61] Liu Y,Zhong Q,Lei H,et al.Study on the antibody against tetrodotoxin made by the immunogen with different carrier proteins[J]. Wei Sheng Yan Jiu,2008;37(2):234-236.
[62]刘燕婷,钟青萍,雷红涛,等.基于不同载体免疫原制备河豚毒素抗体的研究[J].卫生研究,2008,37(2):238～239.
[63] Matsumura K,Fukiya S.Indirect competitive enzyme immunoassay for tetrodotoxin using a biotin-avitin system[J].J AOAC Int,l992,75(5):883-886.
[64] Kreuzer MP,Pravda M,O'Sullivan CK,et al.Novelelectrochemical immunosensors for seafood toxin analysis[J].Toxicon,2002,40(9):1267-1274.
[65] Cheun B,Endo H,Hayashi T,et al.Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning,tetrodotoxin[J].Biosensor Bioelectron,1996,11 (12):1185-1191.
[66]崔建升,陈婧,王芳,王晓辉,魏复盛.微生物传感器测定河豚毒素研究[J].海洋环境科学, 2009, 28(6): 726～728
[67]李德昆,林洪,江洁,等.利用固定化草酸氧化酶法快速检测河豚毒素[J].中国水产科学,2005,12(5):638～642.
[68] Thakur M,Goyal L,Pundir CS.Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase[J].Biochem Bio phys Meth, 2000,44:77-88.
[69] Anderson DM, Kulis DM,Qi YZ, et al. Paralytic shellfish poisoning in southern China [J].Toxicon,1996,34(5):579-590.
[70]王道生,刘乃勤,朱孔平.重度石房蛤毒素中毒1例救治报告[J].医学理论与实践,2009,22(3):329.
[71] Andrew P Negri, Gary J Jones,et al.Sheep Mortality Associated With Paralytic Shellfish Poisons from the Cynobactrium Anabaena Cirinalis[J].Toxicon,1995,33(10):1321-1329.
[72] Smith EA, Grant F, Ferguson CM,et al.Biotransformations of paralytic shellfish toxins by bacteria isolated frombivalve molluscs[J]. Appl Environ Microbiol,2001,67(5):2345-2353.
[73] Van Apeldoorn ME,Van Egmond HP, Speijers GJ, et al. Toxins of cyanobacteria[J].Mol Nutr Food Res,2007,51(1):7-60.
[74] Deeds JR,Landsberg JH, Etheridge SM,et al.Non-traditional vectors for paralytic shellfish poisoning[J].Mar Drugs,2008,6(2):308-348.
[75]王云峰,于仁诚,李钧,等.麻痹性贝类毒素的应用研究[J].海洋科学集刊, 2003,45(6):124～129.
[76] Bordner J,Thiessen WE,Bates HA,et al.The structure of a crystalline derivative of saxitoxin[J].J Am Chem Soc,1975, 97(21):6008-6012.
[77]周名江,李钧,于仁诚,等.赤潮藻毒素研究进展[J].中国海洋药物,1999,18(3):48～54.
[78] Negri A, Stirling D, Quilliam M,et al.Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum[J].Chem Res Toxicol, 2003,16(8):1029-1033.
[79] Yotsu-Yamashita M,Kim YH,Dudley SC Jr,et al.The structure of zetekitoxin AB, a saxitoxin analog from the Panamanian golden frog Atelopus zeteki: a potent sodium-channel blocker[J].Proc Natl Acad Sci U S A,2004,101(13):4346-4351.
[80] Dell'Aversano C,Walter JA,Burton IW,et al.Isolation and structure elucidation of new and unusual saxitoxin analogues from mussels[J].J Nat Prod,2008, 71(9):1518-1523.
[81] Lawrence JF,Maher M,Watson-Wright W.Effect of cooking on the concentration of toxins associated with paralytic shellfish poison in lobster hepatopancreas[J].Toxicon, 1994,32(1):57-64.
[82] Alfonso A,Louzao MC,Vieytes MR,et al.Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples[J].Toxicon,1994, 32(12):1593-1598.
[83] Elizabeth W Maas,Jurgen Thiele1,Caryn Thompson, et al. Modification of an algal culture medium for sustained growth of a saxitoxin-producing isolate of Alexandriumminutum[J].Journal of Applied Pliycology,2000,12:95-98.
[84]王云峰.麻痹性贝毒毒素的制备和活性研究[D].2001.
[85]缪宇平.麻痹性贝毒毒之膝沟藻毒素Gonyautoxins的制备及其测定方法研究[M].2003.
[86] Sunami A,Glaaser IW,Fozzard HA.Structural and gating changes of the sodium channel induced by mutation of aresidue in the upper third of IVS6, creating an external access path for local anesthetics[J].Mol Pharmacol, 2001,59(4):684-691.
[87]宋蔚忠,彭双清,沈勇,等.石房蛤毒素对大鼠心血管和呼吸功能的影响[J].卫生毒理学杂志199913(3):200～201.
[88]陈杰,曹雅明,刘军,等.细胞毒性试验测定麻痹性贝毒素的检测方法[J].中国公共卫生, 2001,11(17):994-995.
[89] Vale C,Alfonso A,Vieytes MR,et al.In vitro and in vivo evaluation of paralytic shellfish poisoning toxin potency and the influence of the pH of extraction[J].Anal Chem,2008,80 (5):1770-1776.
[90] Llewellyn LE.Saxitoxin, a toxic marine natural product that targets a multitude of receptors [J].Nat Prod Rep,2006,23(2):200-222.
[91] Su Z,Sheets M,Ishida H,et al.Saxitoxin blocks L-type Ica[J].J Pharmacol Exp Ther, 2004,308 (1):324-329.
[92] Wang J,Salata JJ,Bennett PB.Saxitoxin is a gating modifier of HERG K+ channels[J].J Gen Physiol,2003,121(6):583-598.
[93] Llewellyn LE,Doyle J.Microtitre plate assay for paralytic shellfish toxins using saxiphilin: gauging the effects of shellfish extract matrices,salts and pH upon assay performance[J]. Toxicon, 2001,39(2-3): 217-224.
[94] Llewellyn LE.Saxitoxin,a toxic marine natural product that targets a multitude of receptors[J]. Nat Prod Rep,2006,23(2):200-222.
[95] Sapse AM,Rothchild R,Rhee K.An ab initio study of the guanidinium groups in saxitoxin[J].J Mol Model,2006,12(2):140-145.
[96] Penzotti JL,Lipkind G,Fozzard HA,et al.Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis[J].Biophys J,2001, 80(2):698-706.
[97] Tanino H,Nakata T,Kaneko T,et al.A stereospecific total synthesis of d,l-saxitoxin[J].J Am Chem Soc,1977, 99(8):2818-2819.
[98] Yotsu-Yamashita M,Kim YH,Dudley SC Jr,et al.The structure of zetekitoxin AB, a saxitoxin analog from the Panamanian golden frog Atelopus zeteki: a potent sodium-channel blocker[J].Proc Natl Acad Sci USA,2004,101(13):4346-4351.
[99] Y Oshima, K Sugino,H Itakura,et al.ToxicMarine Phytoplankton (Eds.: R. Graneli, B. Sundstrom, L.Edler, D. M. Anderson) [J].Elsevier Science, Amsterdam,1990, pp.391–396.
[100] Strichartz GR, Hall S, Magnani B, et al.The potencies of synthetic analogues of saxitoxinand the absolute stereoselectivity of decarbamoyl saxitoxin[J].Toxicon, 1995,33(6): 723-737.
[101] Fleming JJ, Du Bois J.A synthesis of (+)-saxitoxin[J].J Am Chem Soc,2006,128(12): 3926-3927.
[102] Fleming JJ, McReynolds MD, Du Bois J. (+)-saxitoxin: a first and second generation stereoselective synthesis[J].J Am Chem Soc,2007,129(32):9964-9975.
[103] Iwamoto O,Koshino H,Hashizume D,et al.Total synthesis of (-)-decarbamoyloxysaxitoxin [J]. Angew Chem Int Ed Engl,2007,46(45):8625-8628.
[104] Iwamoto O, Shinohara R, Nagasawa K. Total synthesis of (-)- and (+)-decarbamoyloxysaxitoxin and (+)-saxitoxin 1[J].Chem Asian J,2009, 4(2):277-285.
[105] Penzotti JL,Lipkind G,Fozzard HA,et al.Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis[J].Biophys J,2001, 80(2):698-706.
[106] Benton BJ,Keller SA,Spriggs DL,et al.Recovery from the lethal effects of saxitoxin:a therapeutic window for 4-aminopyridine (4-AP) [J].Toxicon,1998,36(4):571-588.
[107] Chanh TC,Hewetson JF.Polyclonal anti-idiotypes induce antibody responses protective against ricin cytotoxicity[J].Immunopharmacology,1993,79(4):681-684.
[108] Hayashi R,Saito H,Okumura M,et al.Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish[J].J Agric Food Chem,2006, 54(2):269-273.
[109] Thibault P, Pleasance S, Laycock MV. Analysis of paralytic shellfish poisons by capillary electrophoresis[J]. J Chromatogr, 1991, 542(2):483-501.
[110] Boyer GL,Goddard GD.High performance liquid chromatography coupled with post-column electrochemical oxidation for the detection of PSP toxins[J].Nat Toxins,1999,7(6):353-359.
[111] Rourke WA,Murphy CJ,Pitcher G,et al.Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue[J]. J AOAC Int,2008,91(3):589-597.
[112] Pleasance S,Ayer SW,Laycock MV,et al.Ionspray mass spectrometry of marine toxins. III.Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry[J]. Rapid Commun Mass Spectrom,1992, 6(1):14-24.
[113] Pleasance S,Thibault P,Kelly J.Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry[J]. J Chromatogr,1992,591(1-2):325-339.
[114] Jaim E,Hummert C,Hess P,et al.Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography[J]. J Chromatogr A,2001,929(1-2):43-49.
[115] Truman P,Lake RJ.Comparison of mouse bioassay and sodium channel cytotoxicity assayfor detectingparalytic shellfish poisoning toxins in shellfish extracts[J]. J AOAC Int,1996,79 (5):1130-1133.
[116] Meadows L,Malhotra JD,Stetzer A,et al.The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit[J].J Neurochem,2001,76(6):1871-1878.
[117] Manger RL,Leja LS,Lee SY,et al.Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitivesodium channels: semiautomated assay for saxitoxins,brevetoxins, andciguatoxins[J].Anal Biochem,1993,214(1):190-194.
[118] Louzao MC,Vieytes MR,Baptista de Sousa JM,et al.A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels[J].Anal Biochem, 2001,289(2):246-250.
[119] Negri A,Llewellyn L.Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxinreceptor assays for paralytic shellfish toxins in crustaceans and molluscs fromtropical North West Australia[J].Toxicon,1998,36(2):283-298.
[120] Krishnan G,Morabito MA,Moczydlowski E.Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin[J].Toxicon,2001,39(2-3):291-301.
[121] Morabito MA,Moczydlowski E.Molecular cloning of bullfrog saxiphilin:a unique relative of the transferrin family that binds saxitoxin[J].Proc Natl Acad Sci USA,1995,92(14):6651.
[122] Chu FS,Fan TS.Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish[J].J Assoc Off Anal Chem,1985,68(1):13-16.
[123] Huot RI,Armstrong DL,Chanh TC.In vitro and in situ inhibition of the sodium channel blocker saxitoxin by monoclonal antibodies[J].J Toxicol Environ Health,1989, 27(3):381-393.
[124] Huot RI,Armstrong DL,Chanh TC.Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin[J].J Clin Invest,1989, 83(6):1821-1826.
[125] Usleber E,Schneider E,Terplan G,et al.Two formats of enzyme immunoassay for the detection of saxitoxin and other paralytic shellfish poisoning toxins[J].Food Addit Contam,1995,12(3):405-413.
[126] Vieytes MR,Cabado AG,Alfonso A,et al.Solid-phase radioreceptor assay for paralytic shellfish toxins[J].Anal Biochem,1993,15,211(1):87-93.
[127] Yong Tang,Hong Wang,Junjian Xiang, et al.A sensitive immunosorbent bio-barcode assay combining PCR with icELISA for detection of gonyautoxin 2/3[J].Analytica Chimica Acta, 2010,657(2):210–214.
[128] Usleber E,Donald M,Straka M,et al.Comparison of enzyme immunoassay and mouse bioassay for determining paralytic shellfish poisoning toxins in shellfish[J].Food Additives & Contaminants,1997,14(2):193-198.
[129] Kawatsu K,Hamano Y,Sugiyama A,et al.Development and application of an enzyme immunoassay based on a monoclonal antibody against gonyautoxin components of paralytic shellfish poisoning toxins[J].J Food Prot,2002,65(8):1304-1308.
[130] Schneider E,Usleber E,Terplan G.Test Strip Enzyme Immunoassay for the Detection of Saxitoxin[J].Food and Agricultural Immunology,1991,3(2):103-104.
[131] Cheun BS,Loughran M,Hayashi T,et al.Use of a channel biosensor for the assay of paralytic shellfish toxins[J].Toxicon,1998,6(10):1371-1381.
[132] Lee HO,Cheun BS,Yoo JS,et al.Application of a channel biosensor for toxicity measurements in cultured lexandrium tamarense[J].J Nat Toxins,2000,9(4):341-348.
[133] Campbell K,Stewart LD,Doucette GJ,et al.Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology[J].Anal Chem,2007,79(15):5906-5914.
[134] Fonfría ES,Vilari?o N,Campbell K,et al.Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes[J].Anal Chem,2007,79(16):6303 -6311.
[135] Katrina Campbella,Anne-Catherine Huetb,Caroline Charlierb, et al.Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins[J]. Journal of Chromatography B, 2009,877 (32):4079-4089.
[136] Pearman WF,Angel SM,Ferry JL,et al.Characterization of the Ag mediated surface-enhanced Raman spectroscopy of saxitoxin[J].Appl Spectrosc,2008,62(7):727-732.
[137]孙丹,袁宏球.石房蛤毒素的研究进展[J].安徽农业科学,2007,35(32): 10204～10224.
[138] Fernández JJ,Candenas ML,Souto ML,et al.Okadaic acid, useful tool for studying cellular processes[J].Curr Med Chem,2002,9(2):229-262.
[139] Lago J,Santaclara F,Vieites JM,et al.Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells[J].Toxicon,2005, 46(5):579-586.
[140] Dickey RW,Bobzin SC,Faulkner DJ,et al.Identification of okadaic acid from a Caribbean dinoflagellate, Prorocentrum concavum[J].Toxicon,1990,28(4):371-377.
[141]谢金镇,刘广发.介核生物分子生物学研究进展[J].台湾海峡,2003,22(3):386～394.
[142] Bravo I,Fernández ML,Ramilo I,et al.Toxin composition of the toxic dinoflagellate Prorocentrum lima isolated from different locations along the Galician coast (NW Spain)[J]. Toxicon,2001,39(10):1537-1545.
[143]杨维东,彭喜春,刘洁生,等.腹泻性贝毒研究现状[J].海洋科学,2005,29(5):66～72.
[144] Barbier M,Amzil Z,Mondeguer F,et al.Okadaic acid and PP2A cellular immunolocalization in Prorocentrum lima (Dinophyceae) [J].Phycologia,1999,38(1):41-46.
[145] Needham J,Hu T,McLachlan J L,et al.Biosynthetic studies of the DSP toxin DTX-4 and an okadaic acid diol ester[J]. J Chem Soc Chem Commum,1995,16:1623-1624.
[146]李青远.海洋生化工具药的研究进展-大田软海绵酸的研究进展[J].中国海洋药物杂志,1995,(1):35～39.
[147] Holmes CF,Luu HA,Carrier F,et al.Inhibition of protein phosphatases-1 and-2A with acanthifolicin. Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition[J].FEBS Lett,1990,270(1-2):216-218.
[148] Nishiwaki S,Fujiki H,Suganuma M,et al.Structure-activity relationship within a series of okadaic acid derivatives[J].Carcinogenesis,1990,11(10):1837-1841.
[149] Paz B,Daranas AH,Cruz PG,et al.Characterisation of okadaic acid related toxins by liquid chromatography coupled with mass spectrometry[J].Toxicon,2007,50(2):225-235.
[150]胡章,马润娣,于立坚.冈田软海绵酸研究进展[J].中国海洋药物杂志,2001,1:46～51.
[151]陈则玲,付云娜,巩宁.腹泻性贝毒及其高效液相色谱检测方法[J].海洋通报,2000,19(1): 73～78.
[152] Fujiki H,Suganuma M.Tumor necrosis factor-alpha, a new tumor promoter, engendered by biochemical studies of okadaic acid[J]. J Biochem,1994,115(1):1-5.
[153] Fernández MT,Zitko V,Gascón S, et al.The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons[J].Life Sci,1991,49(19):157-162.
[154] Matias WG.Transplacental passage of [3H]-okadaic acid in pregnant mice measured by radioactivity and high-performance liquid chromatography[J].Human & Experimental Toxicology,1996,15(3):226-230.
[155] Boudreau RT,Hoskin DW.The use of okadaic acid to elucidate the intracellular role(s) of protein phosphatase 2A:lessons from the mast cell model system[J].Int Immunopharmacol, 2005,5(10):1507-1518.
[156] Harris KA,Oyler GA,Doolittle GM,et al.Okadaic acid induces hyperphosphorylated forms of tau protein in human brain slices[J]. Ann Neurol,1993,3(1):77-87.
[157] Wang SJ, Scavetta R, Lenz HJ, et al. Gene amplification and multidrug resistance induced by the phosphatase-inhibitory tumor promoter, okadaic acid. Carcinogenesis[J]. 1995,16(3):637-641.
[158] Nam KY,Hiro M,Kimura S,et al.Permeability of a non-TPA-type tumor promoter,okadaic acid, through lipid bilayer membrane[J].Carcinogenesis,1990,11(7):1171-1174.
[159] Goto K,Fukuda J,Haneji T.Okadaic acid stimulates apoptosis through expression of Fas receptor and Fas ligand in human oral squamous carcinoma cells[J].Oral Oncol, 2002,38(1):16-22.
[160] Kawai H,Suzuki T,Kobayashi T,et al.Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells[J].Biochim Biophys Acta,2004,693(2):101-110.
[161] Creppy EE,TraoréA,Baudrimont I,et al.Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid[J].Toxicology,2002,181:433-439.
[162]徐年军,范晓,韩丽君,等.藻类抗肿瘤活性物质的研究[J].中国海洋药物,2000,6:30～35.
[163] Fernández JJ,Candenas ML,Souto ML,et al.Okadaic acid, useful tool for studying cellular processes[J].Curr Med Chem,2002,9(2):229-262.
[164] Rundberget T,Sandvik M,Larsen K,et al.Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway,and isolation of okadaic acid and dinophysistoxin-2[J].Toxicon,2007,50(7):960-970.
[165]林永成,周世林.海洋微生物及其代谢产物[M].北京:化学工业出版社,2003.
[166]何水林,郑金贵,王小峰,等.植物次生代谢,功能,调控及其基因工程[J].应用与环境生物学报,2002,8(5):558～563.
[167] Taroncher-O,denburg G,Anderson D M.Identification and characterization of three differentially expressed genes encodings-adenosylhomocysteine hydrolase methiorrine aminopeptidase and a histone-like protein in the toxic Dinofiagellate Alexandrium fundyense[J]. Appl Environ Microbiol,2000,66(5):2105-2112.
[168] Daranas AH,Fernández JJ,Norte M,et al. Biosynthetic studies of the DSP toxin skeleton[J]. Chem Rec,2004, 4(1):1-9.
[169] Dounay AB,Urbanek RA,Sabes SF,et al.Total Synthesis of the Marine Natural Product 7-Deoxy-okadaic Acid:A Potent Inhibitor of Serine/Threonine-Specific Protein Phosphatases [J]. Angew Chem Int Ed Engl,1999,38(15):2258-2262.
[170] Louppis AP, Badeka AV, Katikou P, et al. Determination of okadaic acid, dinophysistoxin-1 and related esters in Greek mussels using HPLC with fluorometric detection, LC-MS/MS and mouse bioassay[J]. Toxicon. 2010,55(4):724-733.
[171]袁祺.舟山渔场及基邻近海域腹泻性贝类毒素的初步研究[J].水产学报,2002,26(6):528～532.
[172] Morton SL, Tindall DR. Determination of okadaic acid content of dinoflagellate cells: a comparison of the HPLC-fluorescent method and two monoclonal antibody ELISA test kits[J]. Toxicon. 1996, 34(8):947-954.
[173] Beatriz Prieto-Simón,Hirotaka Miyachi,Isao Karube,et al.High-sensitive flow-based kinetic exclusion assay for okadaic acid assessment in shellfish samples[J].Biosensor and Bioelectronics, 2010,25 (6):1395-1401.
[174] Elgarch A,Vale P,Rifai S,et al.Detection of diarrheic shellfish poisoning and azaspiracid toxins in Moroccan mussels:comparison of the LC-MS method with the commercial immunoassay kit[J]. Mar Drugs,2008,6(4):587-94.
[175]刘仁沿,梁玉波,张芳,等.中国沿海贝类腹泻性贝毒的酶联免疫分析方法[J].大连海事大学学报,2008,34,(2): 33～36
[176]卢士英,周玉,李岩松,任洪林,霍方珍,王颖,柳增善,于光.大田软海绵酸单克隆抗体ELISA检测方法的建立[J].中国兽医学报,2007,27(3): 336～411
[177]刘仁沿,梁玉波,陈冰君.胶体金免疫层析方法快速检测腹泻性贝毒软海绵酸的初步研究[J].分析试验室,2008,27(7): 26～29
[178]李爱峰,于仁成,李钧.利用蛋白磷酸酶活力抑制法检测牡蛎体内的腹泻性贝毒[J].分析化学,2006,34(3):283～287.
[179] Prassopoulou E, Katikou P, Georgantelis D,et al. Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay,the PP2A inhibition assay and HPLC with fluorimetric detection[J].Toxicon, 2009,53(2):214-227.
[180] Volpe G,Cotroneo E,Moscone D,et al.A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study[J]. Anal Biochem,2009,385(1):50-56.
[181] Campàs M,dela Iglesia P,Le Berre M,et al.Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish[J].Biosensors Bioelectron,2008,24(4):716-722.
[182]朱立平,陈学清.免疫学常用实验方法[M].北京:人民军医出版社,2003.
[183]徐勤惠,荣康泰,恽榴红,等.河豚毒素的新型人工抗原及其高效的抗毒效应[J].免疫学杂志,2003,19(6):424～428.
[184] Kamps-Holtzapple C,Carlin RJ,Sheffield C, et al. Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis[J]. J Immunol Methods,1993, 164(2):245-253.
[185] J.萨姆布鲁克,D W拉塞尔.分子克隆实验指南[M].北京:科学出版社,2002.
[186] Y Zhou, Y S Li, F G Pan, et al.Identification of tetrodotoxin antigens and a monoclonal antibody[J]. Food Chemistry, 2009(112):582-589.
[187]王廷华,李官成.抗体理论与技术[M].北京:科学出版社,2005.
[188]白丽,钱金栿,王晶.用辛酸硫酸胺法提取小鼠腹水和血清中的IgG抗体[J].大理医学院学报,2000,9(4):3～6.
[189]张清华,徐勤惠,王玉霞,等.抗河豚毒素单克隆抗体的制备及其抗毒效应[J].军事医学科学院院刊,2003,27(6):432～434.
[190] Fukiya S,Matsumura K.Active and passive immunization for tetrodotoxin in mice[J]. Toxicon,1992,30(12):1631-1634.
[191] Watabe S,Sato Y,Nakaya M,et al.Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin[J]. Toxicon,1989,27(2):265-268.
[192]崔建洲,申雪艳,宫庆礼,等.高效液相色谱-紫外/荧光检测方法测定河豚毒素[J].色谱, 2006,(24)3:317.
[193] Kreuzer MP,Pravda M,O'Sullivan CK, et al.Novel electrochemical immunosensors for seafood toxin analysis[J]. Toxicon,2002,40(9):1267-1274.
[194] Huang X,Xuan X,Hirata H.Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats[J].J Clin Microbiol,2004, 42(1):351-353.
[195] Chakravarti A,Parthasarathy P,Chakravarti A.Evaluation of a rapid immuno- chromatographic test in the diagnosis of dengue fever[J].Indian J Pathol Microbiol,2003,46(1):127-128.
[196] Guan M,Chen HY,Foo SY, et al.Recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients[J].Clin Diagn Lab Immunol,2004,11(2):287-291.
[197] Huang X,Xuan X,Xu L,et al.Development of an immunochromatographic test with recombinant EMA-2 for the rapid detection of antibodies against Babesia equi in horses[J]. J Clin Microbiol,2004,42(1):359-361.
[198] Smits HL,Abdoel TH,Solera J,et al. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis[J].Clin Diagn Lab Immunol,2003,10(6):1141-1146.
[199] Okame M,Yan H,Akihara S,et al. Evaluation of a newly developed immunochromatographic method for detection of norovirus[J].Kansenshogaku Zasshi, 2003,77(8):637-639.
[200] Toma H,Kobayashi J,Imada Y,et al.Field application and evaluation of a rapid immunochromatographic test for detection of Plasmodium falciparum infection among the inhabitants of Lao PDR[J].Southeast Asian J Trop Med Public Health,2003,34(1):43-47.
[201] Park SK,Lee KW, Hong SH.Development and evaluation of an immunochromatographic kit for the detection of antibody to Plasmodium vivax infection in South Korea[J].Yonsei Med J,2003,44(4):747-750.
[202] Li AD,Li Z,Wang YH,et al.Construction and expression of a single chain antibody mimicing human ovarian cancer antigen CA125[J]. Cell Mol Immunol,2006, (1):59-62.
[203] Li C,Jin NY,Wang HW,et al.Expression of anti-HIV envelope glycoprotein scFv in Pichia pastoris and its biologic activities analysis[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2007, (12):1144-1146.
[204]沈倍奋,陈志南,刘民培.重组抗体[M].北京:科学出版社,2005:347～351.
[205] De Graaf M, Pinedo HM,Oosterhoff D.Pronounced antitumor efficacy by extracellular activation of a doxorubicin-glucuronide prodrug after adenoviral vector-mediated expression of a human antibody-enzyme fusion protein[J]. Hum Gene Ther.2004,15(3): 229-238.
[206] Geng SS, Feng J, Li Y, et al. Binding activity difference of anti-CD20 scFv-Fc fusion protein derived from variable domain exchange[J]. Cell Mol Immunol,2006,3(6):439-443.
[207] Luo D,Mah N,Krantz M, et al.Vl-linker-Vh orientation-dependent expression of singlechain Fv-containing an engineered disulfide-stabilized bond in the framework regions[J].J Biochem,1995,118(4):825-831.
[208] Desplancq D,King DJ,Lawson AD,et al.Multimerization behaviour of single-chain Fv variants for the tumour-binding antibody B72.3[J].Protein Engineering,1994,7(8): 1027-1033.
[209] Asano R,Takemura S,Tsumoto K, et al.Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system[J]. J Biochem, 2000,127(4):673-679.
[210] Nygaard F B,Harlow K W.Heterologous Expression of Soluble,Active Proteins in Escherichia coli:The Human Estrogen Receptor Hormone-Binding Domain as Paradigm[J]. Protein Expression and Purification,2001,21:500-509.
[211] MacDonald L M,Anthony A,Andrew Thompson R C, et al. Expression of Giardia duodenalisβ-Tubulin as a Soluble Protein in Escherichia coli[J].Protein Expression and Purification, 2001,22:25-30.
[212] Dai KS,Zhu HP,Ruan CG.Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ib alpha[J].Thrombosis Research,2003, 109(2-3):137-144.
[213]郭美锦,朱泰承,张明,等.重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响[J].中国生物工程杂志,2007,27(7):7～11.
[214] Mack M, Gruber R, Schmidt S, Riethmüller G, Kufer P. Biologic properties of a bispecific single-chain antibody directed against 17-1A (EpCAM) and CD3: tumor cell-dependent T cell stimulation and cytotoxic activity[J]. J Immunol. 1997, 158(8):3965-3970.
[215]方敏,蒋欣,杨志,等.链间连接肽对单链双特异性抗体生物学活性的影响[J].科学通报,2003,48(18):1912～1918
[2] Noguchi T, Arakawa O. Tetrodotoxin-distribution and acumulation in aquatic organisms, and cases of human intoxication[J]. Mar Drugs,2008, 6(2):220-242.
[3]郑思光,黄朝宗,卢中秋.河豚毒素中毒33例临床分析[J].浙江中西医结合杂志, 2006,16(5):300～301.
[4]马群飞,林升清,王明.烤角片干河豚毒素中毒事件的追踪调查[J].中国卫生监督杂志, 2001,8(4):145～147.
[5]郭柏坤,宫庆礼.河豚毒素的免疫检测进展[J].时珍国医国药,2006,17(4):628～629.
[6]王冠男,谢丽萍,张荣庆.河豚毒素的分布及动物对它的拮抗作用[J].海洋通报, 2007, 26(1):107～113.
[7] Yasumoto T, Nagai H, Yasumura D, et al. Interspecies distribution and possible origin of tetrodotoxin[J]. Ann N Y Acad Sci,1986,479:44-51.
[8]李世平,赵清良,赵强.野生和人工养殖暗纹东方魨不同组织中河豚毒素(TTX)含量的初步研究[J].南京师大学报(自然科学版),1998,21(3):90～94.
[9] Diener M, Christian B, Ahmed MS,et al. Determination of tetrodotoxin and its analogs in the puffer fish Takifugu oblongus from Bangladesh by hydrophilic interaction chromatography and mass-spectrometric detection[J]. Anal Bioanal Chem,2007, 389(6):1997-2002.
[10]李爱峰.液-质联用技术分析海洋生物毒素的研究[D].中国科学院海洋研究所,2005.
[11]吴韶菊,崔建洲,宫庆礼.河豚毒素的微生物起源[J],海洋科学,2005, 29 (10): 81～85.
[12]周玉,王哲.作用于钠通道受体的海产源神经毒素研究概况[J].动物医学进展,2005,26(5):36～40.
[13] Kodama M, Sato S, Ogata T, et al. Tetrodotoxin secreting glands in the skin of puffer fishes[J].Toxicon,1986,24(8):819-829.
[14]张旭东.河豚鱼含毒状祝究进展[J].中国食品卫生杂志,1997,9(1):32～37.
[15] Cheng CA, Hwang DF, Tsai YH, et al. Microflora and tetrodotoxin-producing bacteria in a gastropod, Niotha clathrata[J]. Food Chem Toxicol,1995,33(11):929-934.
[16]陈常英,丁晓琴,潘心富,等.河豚毒素(TTX)及其衍生物的电子结构和构效关系及与石房蛤毒素(STX)的比较研究[J].化学学报,1998,56:109～116.
[17] Kodama M, Noguchi T, Maruyama J, et al. Release of tetrodotoxin and paralytic shellfish poison from puffer liver by RNase[J]. J Biochem,1983,93(1):243-247.
[18] Xu QH, Zhao XN, Wei CH, Rong KT. Immunologic protection of anti-tetrodotoxinvaccines against lethal activities of oral tetrodotoxin challenge in mice[J]. Int Immunopharmacol, 2005,5(7-8):1213-1224.
[19] A Alcaraz,R e Whipple, H R Greeg, B.D. Andresen, et al. Analysis of tetrodotoxin[J]. Forensic Science International, 1999,99(1):35-45.
[20]沈晓书,顾明松,谢剑炜.河豚毒素分析检测方法研究进展[J].军事医学科学院院刊, 2006,30(3):295～298.
[21]桂英爱,王洪军,郝佳.河豚毒素及其代谢产物的研究进展[J].大连水产学院学学报, 2007,22(2):137～141.
[22]代秀梅,庾莉菊,张启明,等.河豚毒素的医药开发前景[J].药品评价, 2008(5) : 230～232.
[23]王小逸,付宏征,林文翰.河豚的化学成分研究[J].中国海洋药物,1999,69(2):4～8.
[24]赵永明,苏晓会,史清文,等.河豚毒素及其类似物的研究概况[J].河北医科大学学报,2007,28(6):474～480.
[25]张凤云,库宝善,姚海燕.河豚毒素单用及与Indoxacarb联合应用的镇痛抗炎作用[J].中国临床康复,2006,10(34):115～118.
[26]符凤英,库宝善,马行,等.河豚毒素与阿司匹林的协同镇痛作用[J].中国临床康复,2006,10(14):100～101.
[27] Daniel M,Schwaetz Z.Experimental study of tetrodotoxin:A long-activating topical anesthetic[J].A m J Ophthalmol,1998,125(4): 481- 487.
[28]杨朝坤,杨双强,谭松涛.河豚毒素停搏液减轻大鼠心肌细胞内钙超载作用的实验研究[J].第三军医大学学报,2005,27(3):192～199.
[29]陈素青,任雷鸣,黄致强.河豚毒素对大鼠和小鼠纳洛酮催促吗啡戒断症状的影响[J].中国药理学与毒理学杂志,2001,15(6):434～440.
[30]刘燕婷,雷红涛,钟青萍.河豚毒素的研究进展[J].食品研究与开发,2008,29(2):156～160.
[31]崔建洲,宫庆礼,顾谦群,等.一种高效制备河豚毒素(TTX)方法的研究[J].高技术通讯, 2005,15(4):89～94.
[32]阚丽丽,郭斌.正交试验法优选超声提取河豚毒素的研究[J].中华中医药学刊,2008,26(6):1274～1275.
[33]梁文京.一起集体河豚毒素食物中毒抢救体会[J].河北医学,2007,13(6):715～717.
[34] Kishi Y,Aratani M,Fukuyama T,et al.Synthetic studies on tetrodotoxin and related compounds.3.A stereospecific synthesis of an equivalent of acetylated tetrodamine[J]. J Am Chem Soc,1972,94(26):9217-9219.
[35] Ohyabu N, Nishikawa T, Isobe M. First asymmetric total synthesis of tetrodotoxin[J]. J Am Chem Soc,2003,125(29):8798-8805.
[36] Hinman A, Du Bois J.A stereoselective synthesis of (-)-tetrodotoxin [J]. J Am Chem Soc,2003,125(38):11510-11511.
[37] Nishikawa T,Asai M,Ohyabu N,et al.Stereocontrolled Synthesis of (-)-5,11- dideoxytetrodotoxin[J].Angewandte Chemie International Edition,1999,38(20):3081-3084.
[38] Sato K, Akai S, Sugita N,et al. Novel and stereocontrolled synthesis of (+/-)-tetrodotoxin from myo-inositol[J]. J Org Chem,2005,70(19):7496-7504.
[39] Urabe D, Nishikawa T, Isobe M. An efficient total synthesis of optically active tetrodotoxin from levoglucosenone[J].Chem Asian J,2006, 7,1(1-2):125-135.
[40] Hwang DF ,Noguchi T.Tetrodotoxin poisoning[J]. Adv Food Nutr Res,2007,52:141-236.
[41] Hamasaki K, Kogure K, Ohwada K. A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culture bioassay in combination with a water-soluble tetrazolium salt[J]. Toxicon.,1996,34(4):490-495.
[42] Davio SR,Fontelo PA.A competitive displacement assay to detect saxitoxin and tetrodotoxin [J]. Anal Biochem,1984,141(1):199-204.
[43] Strong PN,Smith JT, Keana JF. A convenient bioassay for detecting nanomolar concentrations of tetrodotoxin [J]. Toxicon,1973,11(5):433-438.
[44] Cheun B,Endo H,Hayashi T,et al. Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning, tetrodotoxin[J].Biosensor Bioelectron,1996,11 (12):1185-1191.
[45] Nu?ez MT, Fischer S, Jaimovich E.A fluorimetric method to determine tetrodotoxin[J]. Anal Biochem,1976,72:320-325.
[46] Shen XS, Guo L, Gu MS,et al. Rapid determination of tetrodotoxin using microwave assisted alkaline hydrolysis and spectrofluorometric analysis[J].Spectroscopy and spectroscopy analysis,2006;26(12):2264-2267.
[47] Ho B,Yeo DS,Ding JL.A tetrodotoxin neutralizing system in the haemolymph of the horseshoe crab, Carcinoscorpius rotundicauda[J]. Toxicon,1994,32(7):755-762.
[48] Nakamura M,Yasumoto T.Tetrodotoxin derivatives in puffer fish[J].Toxicon,1985, 23(2):271-276.
[49] Ikebuchi J,Suenaga K,Kotoku S,et al. Thin-layer chromatography with flame ionization detection for the determination of tetrodotoxin in biological fluids[J]. J Chromatogr,1988,432:401-406.
[50] Nagashima Y,Nishio S,Noguchi T,et al.Detection of tetrodotoxin by thin-layer chromatography fast atom bombardment mass spectrometry[J].Anal Biochem, 1988,175 (1):258-262.
[51] Kawatsu K,Shibata T,Hamano Y.Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients[J]. Toxicon,1999,37(2):325-333.
[52] O'Leary MA, Schneider JJ, Isbister GK. Use of high performance liquid chromatography to measure tetrodotoxin in serum and urine of poisoned patients[J]. Toxicon, 2004,44(5):549-553.
[53] Shoji Y,Yotsu-Yamashita M,Miyazawa T,et al. Electrospray ionization mass spectrometry of tetrodotoxin and its analogs:liquid chromatography/mass spectrometry, tandem mass spectrometry, and liquid chromatography/tandem mass spectrometry[J].Anal Biochem,2001, 290(1):10-17.
[54]郑雍怡,王彦,张计,等.反相离子对高效液相色谱法测定SD大鼠血浆中河豚毒素的含量[J].分析化学,2008,36(5):588～592.
[55] Hayashida M,Hayakawa H,Wada K,et al.Sensitive determination of tetrodotoxin using column-switching liquid chromatography-mass spectrometry with electrospray ionization in mouse serum [J]. Journal of analytical Toxicology,2004,28(1):46-49.
[56] Pires OR Jr, Sebben A, Schwartz EF,et al.Occurrence of tetrodotoxin and its analogues in the Brazilian frog Brachycephalusephippium(Anura:Brachycephalidae)[J].Toxicon, 2002,40(6):761-766.
[57] Raybould TJ,Bignami GS,Inouye LK, et al.A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples[J].J Clin Lab Anal,1992,6(2):65-72.
[58] Kawatsu K,Shibata T,Hamano Y.Application of immunoaffinity chromatography for detection of tetrodotoxin from urine samples of poisoned patients[J]. Toxicon,1999, 37(2):325-333.
[59] Neagu D,Micheli L,Palleschi G.Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination[J].Anal Bioanal Chem,2006, 385(6):1068-1074.
[60]宫慧芝,杨军.河豚毒素单抗ELISA检测试剂盒的研制[J].中国公共卫生,2005,21(12):1623～1624.
[61] Liu Y,Zhong Q,Lei H,et al.Study on the antibody against tetrodotoxin made by the immunogen with different carrier proteins[J]. Wei Sheng Yan Jiu,2008;37(2):234-236.
[62]刘燕婷,钟青萍,雷红涛,等.基于不同载体免疫原制备河豚毒素抗体的研究[J].卫生研究,2008,37(2):238～239.
[63] Matsumura K,Fukiya S.Indirect competitive enzyme immunoassay for tetrodotoxin using a biotin-avitin system[J].J AOAC Int,l992,75(5):883-886.
[64] Kreuzer MP,Pravda M,O'Sullivan CK,et al.Novelelectrochemical immunosensors for seafood toxin analysis[J].Toxicon,2002,40(9):1267-1274.
[65] Cheun B,Endo H,Hayashi T,et al.Development of an ultra high sensitive tissue biosensor for determination of swellfish poisoning,tetrodotoxin[J].Biosensor Bioelectron,1996,11 (12):1185-1191.
[66]崔建升,陈婧,王芳,王晓辉,魏复盛.微生物传感器测定河豚毒素研究[J].海洋环境科学, 2009, 28(6): 726～728
[67]李德昆,林洪,江洁,等.利用固定化草酸氧化酶法快速检测河豚毒素[J].中国水产科学,2005,12(5):638～642.
[68] Thakur M,Goyal L,Pundir CS.Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase[J].Biochem Bio phys Meth, 2000,44:77-88.
[69] Anderson DM, Kulis DM,Qi YZ, et al. Paralytic shellfish poisoning in southern China [J].Toxicon,1996,34(5):579-590.
[70]王道生,刘乃勤,朱孔平.重度石房蛤毒素中毒1例救治报告[J].医学理论与实践,2009,22(3):329.
[71] Andrew P Negri, Gary J Jones,et al.Sheep Mortality Associated With Paralytic Shellfish Poisons from the Cynobactrium Anabaena Cirinalis[J].Toxicon,1995,33(10):1321-1329.
[72] Smith EA, Grant F, Ferguson CM,et al.Biotransformations of paralytic shellfish toxins by bacteria isolated frombivalve molluscs[J]. Appl Environ Microbiol,2001,67(5):2345-2353.
[73] Van Apeldoorn ME,Van Egmond HP, Speijers GJ, et al. Toxins of cyanobacteria[J].Mol Nutr Food Res,2007,51(1):7-60.
[74] Deeds JR,Landsberg JH, Etheridge SM,et al.Non-traditional vectors for paralytic shellfish poisoning[J].Mar Drugs,2008,6(2):308-348.
[75]王云峰,于仁诚,李钧,等.麻痹性贝类毒素的应用研究[J].海洋科学集刊, 2003,45(6):124～129.
[76] Bordner J,Thiessen WE,Bates HA,et al.The structure of a crystalline derivative of saxitoxin[J].J Am Chem Soc,1975, 97(21):6008-6012.
[77]周名江,李钧,于仁诚,等.赤潮藻毒素研究进展[J].中国海洋药物,1999,18(3):48～54.
[78] Negri A, Stirling D, Quilliam M,et al.Three novel hydroxybenzoate saxitoxin analogues isolated from the dinoflagellate Gymnodinium catenatum[J].Chem Res Toxicol, 2003,16(8):1029-1033.
[79] Yotsu-Yamashita M,Kim YH,Dudley SC Jr,et al.The structure of zetekitoxin AB, a saxitoxin analog from the Panamanian golden frog Atelopus zeteki: a potent sodium-channel blocker[J].Proc Natl Acad Sci U S A,2004,101(13):4346-4351.
[80] Dell'Aversano C,Walter JA,Burton IW,et al.Isolation and structure elucidation of new and unusual saxitoxin analogues from mussels[J].J Nat Prod,2008, 71(9):1518-1523.
[81] Lawrence JF,Maher M,Watson-Wright W.Effect of cooking on the concentration of toxins associated with paralytic shellfish poison in lobster hepatopancreas[J].Toxicon, 1994,32(1):57-64.
[82] Alfonso A,Louzao MC,Vieytes MR,et al.Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples[J].Toxicon,1994, 32(12):1593-1598.
[83] Elizabeth W Maas,Jurgen Thiele1,Caryn Thompson, et al. Modification of an algal culture medium for sustained growth of a saxitoxin-producing isolate of Alexandriumminutum[J].Journal of Applied Pliycology,2000,12:95-98.
[84]王云峰.麻痹性贝毒毒素的制备和活性研究[D].2001.
[85]缪宇平.麻痹性贝毒毒之膝沟藻毒素Gonyautoxins的制备及其测定方法研究[M].2003.
[86] Sunami A,Glaaser IW,Fozzard HA.Structural and gating changes of the sodium channel induced by mutation of aresidue in the upper third of IVS6, creating an external access path for local anesthetics[J].Mol Pharmacol, 2001,59(4):684-691.
[87]宋蔚忠,彭双清,沈勇,等.石房蛤毒素对大鼠心血管和呼吸功能的影响[J].卫生毒理学杂志199913(3):200～201.
[88]陈杰,曹雅明,刘军,等.细胞毒性试验测定麻痹性贝毒素的检测方法[J].中国公共卫生, 2001,11(17):994-995.
[89] Vale C,Alfonso A,Vieytes MR,et al.In vitro and in vivo evaluation of paralytic shellfish poisoning toxin potency and the influence of the pH of extraction[J].Anal Chem,2008,80 (5):1770-1776.
[90] Llewellyn LE.Saxitoxin, a toxic marine natural product that targets a multitude of receptors [J].Nat Prod Rep,2006,23(2):200-222.
[91] Su Z,Sheets M,Ishida H,et al.Saxitoxin blocks L-type Ica[J].J Pharmacol Exp Ther, 2004,308 (1):324-329.
[92] Wang J,Salata JJ,Bennett PB.Saxitoxin is a gating modifier of HERG K+ channels[J].J Gen Physiol,2003,121(6):583-598.
[93] Llewellyn LE,Doyle J.Microtitre plate assay for paralytic shellfish toxins using saxiphilin: gauging the effects of shellfish extract matrices,salts and pH upon assay performance[J]. Toxicon, 2001,39(2-3): 217-224.
[94] Llewellyn LE.Saxitoxin,a toxic marine natural product that targets a multitude of receptors[J]. Nat Prod Rep,2006,23(2):200-222.
[95] Sapse AM,Rothchild R,Rhee K.An ab initio study of the guanidinium groups in saxitoxin[J].J Mol Model,2006,12(2):140-145.
[96] Penzotti JL,Lipkind G,Fozzard HA,et al.Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis[J].Biophys J,2001, 80(2):698-706.
[97] Tanino H,Nakata T,Kaneko T,et al.A stereospecific total synthesis of d,l-saxitoxin[J].J Am Chem Soc,1977, 99(8):2818-2819.
[98] Yotsu-Yamashita M,Kim YH,Dudley SC Jr,et al.The structure of zetekitoxin AB, a saxitoxin analog from the Panamanian golden frog Atelopus zeteki: a potent sodium-channel blocker[J].Proc Natl Acad Sci USA,2004,101(13):4346-4351.
[99] Y Oshima, K Sugino,H Itakura,et al.ToxicMarine Phytoplankton (Eds.: R. Graneli, B. Sundstrom, L.Edler, D. M. Anderson) [J].Elsevier Science, Amsterdam,1990, pp.391–396.
[100] Strichartz GR, Hall S, Magnani B, et al.The potencies of synthetic analogues of saxitoxinand the absolute stereoselectivity of decarbamoyl saxitoxin[J].Toxicon, 1995,33(6): 723-737.
[101] Fleming JJ, Du Bois J.A synthesis of (+)-saxitoxin[J].J Am Chem Soc,2006,128(12): 3926-3927.
[102] Fleming JJ, McReynolds MD, Du Bois J. (+)-saxitoxin: a first and second generation stereoselective synthesis[J].J Am Chem Soc,2007,129(32):9964-9975.
[103] Iwamoto O,Koshino H,Hashizume D,et al.Total synthesis of (-)-decarbamoyloxysaxitoxin [J]. Angew Chem Int Ed Engl,2007,46(45):8625-8628.
[104] Iwamoto O, Shinohara R, Nagasawa K. Total synthesis of (-)- and (+)-decarbamoyloxysaxitoxin and (+)-saxitoxin 1[J].Chem Asian J,2009, 4(2):277-285.
[105] Penzotti JL,Lipkind G,Fozzard HA,et al.Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis[J].Biophys J,2001, 80(2):698-706.
[106] Benton BJ,Keller SA,Spriggs DL,et al.Recovery from the lethal effects of saxitoxin:a therapeutic window for 4-aminopyridine (4-AP) [J].Toxicon,1998,36(4):571-588.
[107] Chanh TC,Hewetson JF.Polyclonal anti-idiotypes induce antibody responses protective against ricin cytotoxicity[J].Immunopharmacology,1993,79(4):681-684.
[108] Hayashi R,Saito H,Okumura M,et al.Cell bioassay for paralytic shellfish poisoning (PSP): comparison with postcolumn derivatization liquid chromatographic analysis and application to the monitoring of PSP in shellfish[J].J Agric Food Chem,2006, 54(2):269-273.
[109] Thibault P, Pleasance S, Laycock MV. Analysis of paralytic shellfish poisons by capillary electrophoresis[J]. J Chromatogr, 1991, 542(2):483-501.
[110] Boyer GL,Goddard GD.High performance liquid chromatography coupled with post-column electrochemical oxidation for the detection of PSP toxins[J].Nat Toxins,1999,7(6):353-359.
[111] Rourke WA,Murphy CJ,Pitcher G,et al.Rapid postcolumn methodology for determination of paralytic shellfish toxins in shellfish tissue[J]. J AOAC Int,2008,91(3):589-597.
[112] Pleasance S,Ayer SW,Laycock MV,et al.Ionspray mass spectrometry of marine toxins. III.Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/mass spectrometry[J]. Rapid Commun Mass Spectrom,1992, 6(1):14-24.
[113] Pleasance S,Thibault P,Kelly J.Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry[J]. J Chromatogr,1992,591(1-2):325-339.
[114] Jaim E,Hummert C,Hess P,et al.Determination of paralytic shellfish poisoning toxins by high-performance ion-exchange chromatography[J]. J Chromatogr A,2001,929(1-2):43-49.
[115] Truman P,Lake RJ.Comparison of mouse bioassay and sodium channel cytotoxicity assayfor detectingparalytic shellfish poisoning toxins in shellfish extracts[J]. J AOAC Int,1996,79 (5):1130-1133.
[116] Meadows L,Malhotra JD,Stetzer A,et al.The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit[J].J Neurochem,2001,76(6):1871-1878.
[117] Manger RL,Leja LS,Lee SY,et al.Tetrazolium-based cell bioassay for neurotoxins active on voltage-sensitivesodium channels: semiautomated assay for saxitoxins,brevetoxins, andciguatoxins[J].Anal Biochem,1993,214(1):190-194.
[118] Louzao MC,Vieytes MR,Baptista de Sousa JM,et al.A fluorimetric method based on changes in membrane potential for screening paralytic shellfish toxins in mussels[J].Anal Biochem, 2001,289(2):246-250.
[119] Negri A,Llewellyn L.Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxinreceptor assays for paralytic shellfish toxins in crustaceans and molluscs fromtropical North West Australia[J].Toxicon,1998,36(2):283-298.
[120] Krishnan G,Morabito MA,Moczydlowski E.Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin[J].Toxicon,2001,39(2-3):291-301.
[121] Morabito MA,Moczydlowski E.Molecular cloning of bullfrog saxiphilin:a unique relative of the transferrin family that binds saxitoxin[J].Proc Natl Acad Sci USA,1995,92(14):6651.
[122] Chu FS,Fan TS.Indirect enzyme-linked immunosorbent assay for saxitoxin in shellfish[J].J Assoc Off Anal Chem,1985,68(1):13-16.
[123] Huot RI,Armstrong DL,Chanh TC.In vitro and in situ inhibition of the sodium channel blocker saxitoxin by monoclonal antibodies[J].J Toxicol Environ Health,1989, 27(3):381-393.
[124] Huot RI,Armstrong DL,Chanh TC.Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin[J].J Clin Invest,1989, 83(6):1821-1826.
[125] Usleber E,Schneider E,Terplan G,et al.Two formats of enzyme immunoassay for the detection of saxitoxin and other paralytic shellfish poisoning toxins[J].Food Addit Contam,1995,12(3):405-413.
[126] Vieytes MR,Cabado AG,Alfonso A,et al.Solid-phase radioreceptor assay for paralytic shellfish toxins[J].Anal Biochem,1993,15,211(1):87-93.
[127] Yong Tang,Hong Wang,Junjian Xiang, et al.A sensitive immunosorbent bio-barcode assay combining PCR with icELISA for detection of gonyautoxin 2/3[J].Analytica Chimica Acta, 2010,657(2):210–214.
[128] Usleber E,Donald M,Straka M,et al.Comparison of enzyme immunoassay and mouse bioassay for determining paralytic shellfish poisoning toxins in shellfish[J].Food Additives & Contaminants,1997,14(2):193-198.
[129] Kawatsu K,Hamano Y,Sugiyama A,et al.Development and application of an enzyme immunoassay based on a monoclonal antibody against gonyautoxin components of paralytic shellfish poisoning toxins[J].J Food Prot,2002,65(8):1304-1308.
[130] Schneider E,Usleber E,Terplan G.Test Strip Enzyme Immunoassay for the Detection of Saxitoxin[J].Food and Agricultural Immunology,1991,3(2):103-104.
[131] Cheun BS,Loughran M,Hayashi T,et al.Use of a channel biosensor for the assay of paralytic shellfish toxins[J].Toxicon,1998,6(10):1371-1381.
[132] Lee HO,Cheun BS,Yoo JS,et al.Application of a channel biosensor for toxicity measurements in cultured lexandrium tamarense[J].J Nat Toxins,2000,9(4):341-348.
[133] Campbell K,Stewart LD,Doucette GJ,et al.Assessment of specific binding proteins suitable for the detection of paralytic shellfish poisons using optical biosensor technology[J].Anal Chem,2007,79(15):5906-5914.
[134] Fonfría ES,Vilari?o N,Campbell K,et al.Paralytic shellfish poisoning detection by surface plasmon resonance-based biosensors in shellfish matrixes[J].Anal Chem,2007,79(16):6303 -6311.
[135] Katrina Campbella,Anne-Catherine Huetb,Caroline Charlierb, et al.Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins[J]. Journal of Chromatography B, 2009,877 (32):4079-4089.
[136] Pearman WF,Angel SM,Ferry JL,et al.Characterization of the Ag mediated surface-enhanced Raman spectroscopy of saxitoxin[J].Appl Spectrosc,2008,62(7):727-732.
[137]孙丹,袁宏球.石房蛤毒素的研究进展[J].安徽农业科学,2007,35(32): 10204～10224.
[138] Fernández JJ,Candenas ML,Souto ML,et al.Okadaic acid, useful tool for studying cellular processes[J].Curr Med Chem,2002,9(2):229-262.
[139] Lago J,Santaclara F,Vieites JM,et al.Collapse of mitochondrial membrane potential and caspases activation are early events in okadaic acid-treated Caco-2 cells[J].Toxicon,2005, 46(5):579-586.
[140] Dickey RW,Bobzin SC,Faulkner DJ,et al.Identification of okadaic acid from a Caribbean dinoflagellate, Prorocentrum concavum[J].Toxicon,1990,28(4):371-377.
[141]谢金镇,刘广发.介核生物分子生物学研究进展[J].台湾海峡,2003,22(3):386～394.
[142] Bravo I,Fernández ML,Ramilo I,et al.Toxin composition of the toxic dinoflagellate Prorocentrum lima isolated from different locations along the Galician coast (NW Spain)[J]. Toxicon,2001,39(10):1537-1545.
[143]杨维东,彭喜春,刘洁生,等.腹泻性贝毒研究现状[J].海洋科学,2005,29(5):66～72.
[144] Barbier M,Amzil Z,Mondeguer F,et al.Okadaic acid and PP2A cellular immunolocalization in Prorocentrum lima (Dinophyceae) [J].Phycologia,1999,38(1):41-46.
[145] Needham J,Hu T,McLachlan J L,et al.Biosynthetic studies of the DSP toxin DTX-4 and an okadaic acid diol ester[J]. J Chem Soc Chem Commum,1995,16:1623-1624.
[146]李青远.海洋生化工具药的研究进展-大田软海绵酸的研究进展[J].中国海洋药物杂志,1995,(1):35～39.
[147] Holmes CF,Luu HA,Carrier F,et al.Inhibition of protein phosphatases-1 and-2A with acanthifolicin. Comparison with diarrhetic shellfish toxins and identification of a region on okadaic acid important for phosphatase inhibition[J].FEBS Lett,1990,270(1-2):216-218.
[148] Nishiwaki S,Fujiki H,Suganuma M,et al.Structure-activity relationship within a series of okadaic acid derivatives[J].Carcinogenesis,1990,11(10):1837-1841.
[149] Paz B,Daranas AH,Cruz PG,et al.Characterisation of okadaic acid related toxins by liquid chromatography coupled with mass spectrometry[J].Toxicon,2007,50(2):225-235.
[150]胡章,马润娣,于立坚.冈田软海绵酸研究进展[J].中国海洋药物杂志,2001,1:46～51.
[151]陈则玲,付云娜,巩宁.腹泻性贝毒及其高效液相色谱检测方法[J].海洋通报,2000,19(1): 73～78.
[152] Fujiki H,Suganuma M.Tumor necrosis factor-alpha, a new tumor promoter, engendered by biochemical studies of okadaic acid[J]. J Biochem,1994,115(1):1-5.
[153] Fernández MT,Zitko V,Gascón S, et al.The marine toxin okadaic acid is a potent neurotoxin for cultured cerebellar neurons[J].Life Sci,1991,49(19):157-162.
[154] Matias WG.Transplacental passage of [3H]-okadaic acid in pregnant mice measured by radioactivity and high-performance liquid chromatography[J].Human & Experimental Toxicology,1996,15(3):226-230.
[155] Boudreau RT,Hoskin DW.The use of okadaic acid to elucidate the intracellular role(s) of protein phosphatase 2A:lessons from the mast cell model system[J].Int Immunopharmacol, 2005,5(10):1507-1518.
[156] Harris KA,Oyler GA,Doolittle GM,et al.Okadaic acid induces hyperphosphorylated forms of tau protein in human brain slices[J]. Ann Neurol,1993,3(1):77-87.
[157] Wang SJ, Scavetta R, Lenz HJ, et al. Gene amplification and multidrug resistance induced by the phosphatase-inhibitory tumor promoter, okadaic acid. Carcinogenesis[J]. 1995,16(3):637-641.
[158] Nam KY,Hiro M,Kimura S,et al.Permeability of a non-TPA-type tumor promoter,okadaic acid, through lipid bilayer membrane[J].Carcinogenesis,1990,11(7):1171-1174.
[159] Goto K,Fukuda J,Haneji T.Okadaic acid stimulates apoptosis through expression of Fas receptor and Fas ligand in human oral squamous carcinoma cells[J].Oral Oncol, 2002,38(1):16-22.
[160] Kawai H,Suzuki T,Kobayashi T,et al.Simultaneous imaging of initiator/effector caspase activity and mitochondrial membrane potential during cell death in living HeLa cells[J].Biochim Biophys Acta,2004,693(2):101-110.
[161] Creppy EE,TraoréA,Baudrimont I,et al.Recent advances in the study of epigenetic effects induced by the phycotoxin okadaic acid[J].Toxicology,2002,181:433-439.
[162]徐年军,范晓,韩丽君,等.藻类抗肿瘤活性物质的研究[J].中国海洋药物,2000,6:30～35.
[163] Fernández JJ,Candenas ML,Souto ML,et al.Okadaic acid, useful tool for studying cellular processes[J].Curr Med Chem,2002,9(2):229-262.
[164] Rundberget T,Sandvik M,Larsen K,et al.Extraction of microalgal toxins by large-scale pumping of seawater in Spain and Norway,and isolation of okadaic acid and dinophysistoxin-2[J].Toxicon,2007,50(7):960-970.
[165]林永成,周世林.海洋微生物及其代谢产物[M].北京:化学工业出版社,2003.
[166]何水林,郑金贵,王小峰,等.植物次生代谢,功能,调控及其基因工程[J].应用与环境生物学报,2002,8(5):558～563.
[167] Taroncher-O,denburg G,Anderson D M.Identification and characterization of three differentially expressed genes encodings-adenosylhomocysteine hydrolase methiorrine aminopeptidase and a histone-like protein in the toxic Dinofiagellate Alexandrium fundyense[J]. Appl Environ Microbiol,2000,66(5):2105-2112.
[168] Daranas AH,Fernández JJ,Norte M,et al. Biosynthetic studies of the DSP toxin skeleton[J]. Chem Rec,2004, 4(1):1-9.
[169] Dounay AB,Urbanek RA,Sabes SF,et al.Total Synthesis of the Marine Natural Product 7-Deoxy-okadaic Acid:A Potent Inhibitor of Serine/Threonine-Specific Protein Phosphatases [J]. Angew Chem Int Ed Engl,1999,38(15):2258-2262.
[170] Louppis AP, Badeka AV, Katikou P, et al. Determination of okadaic acid, dinophysistoxin-1 and related esters in Greek mussels using HPLC with fluorometric detection, LC-MS/MS and mouse bioassay[J]. Toxicon. 2010,55(4):724-733.
[171]袁祺.舟山渔场及基邻近海域腹泻性贝类毒素的初步研究[J].水产学报,2002,26(6):528～532.
[172] Morton SL, Tindall DR. Determination of okadaic acid content of dinoflagellate cells: a comparison of the HPLC-fluorescent method and two monoclonal antibody ELISA test kits[J]. Toxicon. 1996, 34(8):947-954.
[173] Beatriz Prieto-Simón,Hirotaka Miyachi,Isao Karube,et al.High-sensitive flow-based kinetic exclusion assay for okadaic acid assessment in shellfish samples[J].Biosensor and Bioelectronics, 2010,25 (6):1395-1401.
[174] Elgarch A,Vale P,Rifai S,et al.Detection of diarrheic shellfish poisoning and azaspiracid toxins in Moroccan mussels:comparison of the LC-MS method with the commercial immunoassay kit[J]. Mar Drugs,2008,6(4):587-94.
[175]刘仁沿,梁玉波,张芳,等.中国沿海贝类腹泻性贝毒的酶联免疫分析方法[J].大连海事大学学报,2008,34,(2): 33～36
[176]卢士英,周玉,李岩松,任洪林,霍方珍,王颖,柳增善,于光.大田软海绵酸单克隆抗体ELISA检测方法的建立[J].中国兽医学报,2007,27(3): 336～411
[177]刘仁沿,梁玉波,陈冰君.胶体金免疫层析方法快速检测腹泻性贝毒软海绵酸的初步研究[J].分析试验室,2008,27(7): 26～29
[178]李爱峰,于仁成,李钧.利用蛋白磷酸酶活力抑制法检测牡蛎体内的腹泻性贝毒[J].分析化学,2006,34(3):283～287.
[179] Prassopoulou E, Katikou P, Georgantelis D,et al. Detection of okadaic acid and related esters in mussels during diarrhetic shellfish poisoning (DSP) episodes in Greece using the mouse bioassay,the PP2A inhibition assay and HPLC with fluorimetric detection[J].Toxicon, 2009,53(2):214-227.
[180] Volpe G,Cotroneo E,Moscone D,et al.A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study[J]. Anal Biochem,2009,385(1):50-56.
[181] Campàs M,dela Iglesia P,Le Berre M,et al.Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish[J].Biosensors Bioelectron,2008,24(4):716-722.
[182]朱立平,陈学清.免疫学常用实验方法[M].北京:人民军医出版社,2003.
[183]徐勤惠,荣康泰,恽榴红,等.河豚毒素的新型人工抗原及其高效的抗毒效应[J].免疫学杂志,2003,19(6):424～428.
[184] Kamps-Holtzapple C,Carlin RJ,Sheffield C, et al. Analysis of hapten-carrier protein conjugates by nondenaturing gel electrophoresis[J]. J Immunol Methods,1993, 164(2):245-253.
[185] J.萨姆布鲁克,D W拉塞尔.分子克隆实验指南[M].北京:科学出版社,2002.
[186] Y Zhou, Y S Li, F G Pan, et al.Identification of tetrodotoxin antigens and a monoclonal antibody[J]. Food Chemistry, 2009(112):582-589.
[187]王廷华,李官成.抗体理论与技术[M].北京:科学出版社,2005.
[188]白丽,钱金栿,王晶.用辛酸硫酸胺法提取小鼠腹水和血清中的IgG抗体[J].大理医学院学报,2000,9(4):3～6.
[189]张清华,徐勤惠,王玉霞,等.抗河豚毒素单克隆抗体的制备及其抗毒效应[J].军事医学科学院院刊,2003,27(6):432～434.
[190] Fukiya S,Matsumura K.Active and passive immunization for tetrodotoxin in mice[J]. Toxicon,1992,30(12):1631-1634.
[191] Watabe S,Sato Y,Nakaya M,et al.Monoclonal antibody raised against tetrodonic acid, a derivative of tetrodotoxin[J]. Toxicon,1989,27(2):265-268.
[192]崔建洲,申雪艳,宫庆礼,等.高效液相色谱-紫外/荧光检测方法测定河豚毒素[J].色谱, 2006,(24)3:317.
[193] Kreuzer MP,Pravda M,O'Sullivan CK, et al.Novel electrochemical immunosensors for seafood toxin analysis[J]. Toxicon,2002,40(9):1267-1274.
[194] Huang X,Xuan X,Hirata H.Rapid immunochromatographic test using recombinant SAG2 for detection of antibodies against Toxoplasma gondii in cats[J].J Clin Microbiol,2004, 42(1):351-353.
[195] Chakravarti A,Parthasarathy P,Chakravarti A.Evaluation of a rapid immuno- chromatographic test in the diagnosis of dengue fever[J].Indian J Pathol Microbiol,2003,46(1):127-128.
[196] Guan M,Chen HY,Foo SY, et al.Recombinant protein-based enzyme-linked immunosorbent assay and immunochromatographic tests for detection of immunoglobulin G antibodies to severe acute respiratory syndrome (SARS) coronavirus in SARS patients[J].Clin Diagn Lab Immunol,2004,11(2):287-291.
[197] Huang X,Xuan X,Xu L,et al.Development of an immunochromatographic test with recombinant EMA-2 for the rapid detection of antibodies against Babesia equi in horses[J]. J Clin Microbiol,2004,42(1):359-361.
[198] Smits HL,Abdoel TH,Solera J,et al. Immunochromatographic Brucella-specific immunoglobulin M and G lateral flow assays for rapid serodiagnosis of human brucellosis[J].Clin Diagn Lab Immunol,2003,10(6):1141-1146.
[199] Okame M,Yan H,Akihara S,et al. Evaluation of a newly developed immunochromatographic method for detection of norovirus[J].Kansenshogaku Zasshi, 2003,77(8):637-639.
[200] Toma H,Kobayashi J,Imada Y,et al.Field application and evaluation of a rapid immunochromatographic test for detection of Plasmodium falciparum infection among the inhabitants of Lao PDR[J].Southeast Asian J Trop Med Public Health,2003,34(1):43-47.
[201] Park SK,Lee KW, Hong SH.Development and evaluation of an immunochromatographic kit for the detection of antibody to Plasmodium vivax infection in South Korea[J].Yonsei Med J,2003,44(4):747-750.
[202] Li AD,Li Z,Wang YH,et al.Construction and expression of a single chain antibody mimicing human ovarian cancer antigen CA125[J]. Cell Mol Immunol,2006, (1):59-62.
[203] Li C,Jin NY,Wang HW,et al.Expression of anti-HIV envelope glycoprotein scFv in Pichia pastoris and its biologic activities analysis[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2007, (12):1144-1146.
[204]沈倍奋,陈志南,刘民培.重组抗体[M].北京:科学出版社,2005:347～351.
[205] De Graaf M, Pinedo HM,Oosterhoff D.Pronounced antitumor efficacy by extracellular activation of a doxorubicin-glucuronide prodrug after adenoviral vector-mediated expression of a human antibody-enzyme fusion protein[J]. Hum Gene Ther.2004,15(3): 229-238.
[206] Geng SS, Feng J, Li Y, et al. Binding activity difference of anti-CD20 scFv-Fc fusion protein derived from variable domain exchange[J]. Cell Mol Immunol,2006,3(6):439-443.
[207] Luo D,Mah N,Krantz M, et al.Vl-linker-Vh orientation-dependent expression of singlechain Fv-containing an engineered disulfide-stabilized bond in the framework regions[J].J Biochem,1995,118(4):825-831.
[208] Desplancq D,King DJ,Lawson AD,et al.Multimerization behaviour of single-chain Fv variants for the tumour-binding antibody B72.3[J].Protein Engineering,1994,7(8): 1027-1033.
[209] Asano R,Takemura S,Tsumoto K, et al.Functional construction of the anti-mucin core protein (MUC1) antibody MUSE11 variable regions in a bacterial expression system[J]. J Biochem, 2000,127(4):673-679.
[210] Nygaard F B,Harlow K W.Heterologous Expression of Soluble,Active Proteins in Escherichia coli:The Human Estrogen Receptor Hormone-Binding Domain as Paradigm[J]. Protein Expression and Purification,2001,21:500-509.
[211] MacDonald L M,Anthony A,Andrew Thompson R C, et al. Expression of Giardia duodenalisβ-Tubulin as a Soluble Protein in Escherichia coli[J].Protein Expression and Purification, 2001,22:25-30.
[212] Dai KS,Zhu HP,Ruan CG.Generation and characterization of recombinant single chain Fv antibody that recognizes platelet glycoprotein Ib alpha[J].Thrombosis Research,2003, 109(2-3):137-144.
[213]郭美锦,朱泰承,张明,等.重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响[J].中国生物工程杂志,2007,27(7):7～11.
[214] Mack M, Gruber R, Schmidt S, Riethmüller G, Kufer P. Biologic properties of a bispecific single-chain antibody directed against 17-1A (EpCAM) and CD3: tumor cell-dependent T cell stimulation and cytotoxic activity[J]. J Immunol. 1997, 158(8):3965-3970.
[215]方敏,蒋欣,杨志,等.链间连接肽对单链双特异性抗体生物学活性的影响[J].科学通报,2003,48(18):1912～1918
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