微囊藻毒素LR免疫原及包被抗原的合成与鉴定
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摘要
微囊藻毒素LR(MC-LR)分子量小于1 000 u,为半抗原,需要与载体蛋白偶联后才能刺激免疫应答用于抗体制备。本研究利用碳二亚胺法将MC-LR分别与钥孔血蓝蛋白(KLH)和牛血清白蛋白(BSA)进行偶联,获得免疫抗原(MC-KLH)和包被抗原(MC-BSA)。采用紫外扫描光谱、SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)、基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)等3种方法对2种人工抗原进行鉴定,并对小鼠进行免疫。用间接非竞争酶联免疫吸附测定法(ELISA)和竞争ELISA测定了抗血清效价和敏感度。结果表明,紫外扫描光谱法可观察到2种人工抗原的紫外吸收谱与载体蛋白相比发生改变。通过SDS-PAGE法可以观察到MC-BSA分子量比BSA增大。另外质谱法测定的MC-BSA的相对分子量为76 515.84,计算得到的MC-LR与BSA的偶联比为10∶1。MC-KLH免疫小鼠后,抗血清效价最高达到1∶32 000倍,MC-LR对鼠多抗血清的抑制中浓度(I_(50))为0.53μg/mL。成功合成了MC-LR的免疫原和包被原为其单克隆抗体的制备奠定了基础。还比较了3种鉴定方法的适用性和优缺点,为其他人工抗原的鉴定方法提供了有益的参考。
引文
[1]闫海,潘纲,张明明,等.微囊藻毒素的提取和提纯研究[J].环境科学学报,2004,24(2):355-359.
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[5]刘媛,Tuomas H,刘贤金,等.基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定[J].中国农业科学,2012,45(2):330-337.
[6]顾丽丽.ELISA试剂盒法测定水中LR型微囊藻毒素[J].化学分析计量,2013,22(1):97-99.
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[12]Aranda-Rodriguez R,Kubwabo C,Benoit F M.Extraction of 15 microcystins and nodularin using immunoaffinity columns[J].Toxicon,2003,42(6):587-599.
[13]Ward C J,Beattie K A,Lee E Y,et al.Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria:comparisons with high-performance liquid chromatographic analysis for microcystins[J].Fems Microbiology Letters,1997,153(2):465-473.
[14]Heresztyn T,Nicholson B C.Determination of cyanobacterial hepatotoxins directly in water using a protein phosphatase inhibition assay[J].Water Research,2001,35(13):3049-3056.
[15]Kiviranta J,Sivonen K,Niemel? S I,et al.Detection of toxicity of cyanobacteria by Artemia salina bioassay[J].Environmental Toxicology & Water Quality,1991,6(4):423-436.
[16]Mcelhiney J,Lawton L A.Detection of the cyanobacterial hepatotoxins microcystins[J].Toxicology & Applied Pharmacology,2005,203(3):219-230.
[17]Yu F Y,Liu B H,Chou H N,et al.Development of a sensitive ELISA for the determination of microcystins in algae[J].Journal of Agricultural & Food Chemistry,2002,50(15):4176-4182.
[18]周思祥,刘福成.农药人工抗原的合成研究进展[J].农药,2005,44(8):337-341.
[19]Metcalf J S,Bell S G,Codd G A.Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin[J].Water Research,2000,34(10):2761-2769.
[20]Zeck A,Eikenberg A,Weller M G,et al.Highly sensitive immunoassay based on a monoclonal antibody specific for[4-arginine]microcystins[J].Analytica Chimica Acta,2001,441(1):1-13.
[21]盛建武,何苗,宋保栋,等.微囊藻毒素-LR完全抗原的设计及制备[J].环境科学,2005,26(3):33-37.
[2]Humpage A R,Falconer I R.Oral toxicity of the cyanobacterial toxin cylindrospermopsin in male Swiss albino mice:determination of no observed adverse effect level for deriving a drinking water guideline value[J].Environmental Toxicology,2003,18(2):94-103.
[3]Blahova L,Marsalek O B,Sejnohova L,et al.The first occurrence of the cyanobacterial alkaloid toxin cylindrospermopsin in the Czech Republic as determined by immunochemical and LC/MS methods[J].Toxicon,2009,53(5):519-524.
[4]Meng G M,Sun Y,Fu W Y,et al.Microcystin-LR induces cytoskeleton system reorganization through hyperphosphorylation of tau and HSP27 via PP2A inhibition and subsequent activation of the p38 MAPK signaling pathway in neuroendocrine (PC12) cells[J].Toxicology,2011,290(2/3):218-229.
[5]刘媛,Tuomas H,刘贤金,等.基于磁珠和时间分辨荧光免疫分析的微囊藻毒素LR单链抗体筛选与鉴定[J].中国农业科学,2012,45(2):330-337.
[6]顾丽丽.ELISA试剂盒法测定水中LR型微囊藻毒素[J].化学分析计量,2013,22(1):97-99.
[7]Rapala J,Erkomaa K,Kukkonen J,et al.Detection of microcystins with protein phosphatase inhibition assay,high-performance liquid chromatography-UV detection and enzyme-linked immunosorbent assay:comparison of methods[J].Analytica Chimica Acta,2002,466(2):213-231.
[8]Dahlmann J,Budakowski W R,Luckas B.Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins[J].Journal of Chromatography A,2003,994(1/2):45-57.
[9]Spoof L,Vesterkvist P,Lindholm T,et al.Screening for cyanobacterial hepatotoxins,microcystins and nodularin in environmental water samples by reversed-phase liquid chromatography-electrospray ionisation mass spectrometry[J].Journal of Chromatography A,2003,1020(1):105-119.
[10]Fischer W J,Garthwaite I,Miles C O,et al.Congener-independent immunoassay for microcystins and nodularins[J].Environmental Science & Technology,2001,35(24):4849-4856.
[11]Tsutsumi T,Nagata S,Hasegawa A,et al.Immunoaffinity column as clean-up tool for determination of trace amounts of microcystins in tap water[J].Food & Chemical Toxicology,2000,38(7):593-597.
[12]Aranda-Rodriguez R,Kubwabo C,Benoit F M.Extraction of 15 microcystins and nodularin using immunoaffinity columns[J].Toxicon,2003,42(6):587-599.
[13]Ward C J,Beattie K A,Lee E Y,et al.Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria:comparisons with high-performance liquid chromatographic analysis for microcystins[J].Fems Microbiology Letters,1997,153(2):465-473.
[14]Heresztyn T,Nicholson B C.Determination of cyanobacterial hepatotoxins directly in water using a protein phosphatase inhibition assay[J].Water Research,2001,35(13):3049-3056.
[15]Kiviranta J,Sivonen K,Niemel? S I,et al.Detection of toxicity of cyanobacteria by Artemia salina bioassay[J].Environmental Toxicology & Water Quality,1991,6(4):423-436.
[16]Mcelhiney J,Lawton L A.Detection of the cyanobacterial hepatotoxins microcystins[J].Toxicology & Applied Pharmacology,2005,203(3):219-230.
[17]Yu F Y,Liu B H,Chou H N,et al.Development of a sensitive ELISA for the determination of microcystins in algae[J].Journal of Agricultural & Food Chemistry,2002,50(15):4176-4182.
[18]周思祥,刘福成.农药人工抗原的合成研究进展[J].农药,2005,44(8):337-341.
[19]Metcalf J S,Bell S G,Codd G A.Production of novel polyclonal antibodies against the cyanobacterial toxin microcystin-LR and their application for the detection and quantification of microcystins and nodularin[J].Water Research,2000,34(10):2761-2769.
[20]Zeck A,Eikenberg A,Weller M G,et al.Highly sensitive immunoassay based on a monoclonal antibody specific for[4-arginine]microcystins[J].Analytica Chimica Acta,2001,441(1):1-13.
[21]盛建武,何苗,宋保栋,等.微囊藻毒素-LR完全抗原的设计及制备[J].环境科学,2005,26(3):33-37.