【作者】 张卓；

【导师】 江天久；

【作者基本信息】 暨南大学 ， 动物学， 2018， 硕士

【摘要】 膝沟藻毒素1～4(gonyautoxin 2&3,gonyautoxin 1&4;GTX2,3,GTX1,4)是氨基甲酸酯类麻痹性贝类毒素(paralytic shellfish poisoning,PSP)的主要成分,是我国南部沿海地区染毒贝类及有害藻华的主要毒素,对我国沿海地区贝类食用安全及消费者生命健康产生严重隐患。因此,需要建立一种快速便捷的现场检测手段以利于贝类毒素的监测和管理。本研究运用单克隆抗体和胶体金免疫层析技术针对膝沟藻毒素GTX1～4开展研究。主要研究内容包括:(1)利用甲醛法,分别合成作为免疫抗原GTX1～4-BSA和包被抗原GTX1～4-KLH,并对合成的抗原进行鉴定;(2)以GTX1～4-BSA作为免疫原对BALB/c小鼠进行免疫,利用细胞融合技术筛选能够产生抗GTX1～4的细胞株,并对腹水型抗体进行鉴定;(3)以腹水型抗体为原料,通过优化实验条件,对麻痹性贝毒GTX1～4胶体金试纸条进行初步探索。实验结果如下:成功制备了GTX1～4的完全抗原,完全抗原的偶联比(GTX1～4:BSA)在3～35:1之间,其中GTX1～4-BSA的浓度为5.09 mg/mL,GTX1～4-KLH的浓度为3.75 mg/mL。利用间接ELISA法,成功筛选出三株能稳定分泌抗GTX1～4的杂交瘤细胞,分别命名为1-A4、5-A11和5-E11。三株杂交瘤细胞所制备腹水的效价分别为1:1.02×106、1:1.02×106和1:2.04×106。1-A4、5-A11腹水单抗经过亚型鉴定重链均为IgG1,5-E11重链为Ig G2b,三株细胞腹水单抗轻链均为κ链。竞争ELISA法结果显示,三株细胞所产腹水单抗均能与GTX1～4-KLH包被抗原发生特异性反应,其中5-E11单抗IC50最低为6.25μg/mL。交叉反应检测结果显示5-E11单克隆抗体与常见同系海洋毒素不发生交叉反应。对胶体金试纸条的初步研究结果表明,当柠檬酸三钠浓度为5%,反应时间为10min时,能够得到粒径集中,单一分散性好的胶体金溶液;当胶体金和腹水单抗偶联时间为20 min,抗体溶液稀释5倍时,能够达到最好的偶联效果。最佳金标蛋白量为5μg,最佳p H值为8.0,硝酸纤维素膜在24℃下需干燥24 h能达到稳定状态。稳定性实验结果显示常温下在三个月内试纸条基本性能不变。在此基础上初步建立了胶体金免疫层析试纸条检测GTX1～4的方法,其对GTX1-4的最低检测限为12.5μg/m L。更多还原

【Abstract】 Gonyautoxin 1～4(gonyautoxin 2&3,gonyautoxin 1&4;GTX2,3,GTX1,4)which was the main component of carbamate paralytic shellfish toxin(paralytic shellfish poisoning,PSP),and one of the major shellfish toxins in the southern coastal areas of China,had brought serious security risks to seafood products and consumer health.Therefore,it was necessary to establish a fast and convenient on-site detection method to ensure the safety of consumers.Therefore,it is necessary to establish a fast and convenient on-site detection method to facilitate the monitoring and management of shellfish toxins.In this study,monoclonal antibodies and colloidal gold immunochromatography were used to study the toxin GTX1～4.The main contents of this study were as follows:(1)using the formaldehyde method,the immunogen GTX1～4-BSA and the inclusion antigen GTX1～4-KLH were synthesized and the synthesized antigens were identified;(2)GTX1～4-BSA was used as the immunogen to BALB/c.The mice were immunized and screened by cell fusion technology to produce anti GTX1～4 cell lines and identification of ascitic antibodies.(3)a preliminary exploration was carried out on the colloid gold paper strip of paralytic shellfish by optimizing the experimental conditions by using ascitic antibody as the raw material.The experimental results are as follows:The complete antigen of GTX1～4 was successfully prepared,and the coupling ratio of complete antigen(GTX1～4:BSA)was between 3～35:1,in which the concentration of GTX1～4-BSA was5.09 mg/mL and the concentration of GTX1～4-KLH was 3.75 mg/m L.By indirect ELISA method,three strains of hybridoma cells capable of stabilizing the secretion of anti GTX1～4 hybridoma were successfully screened,named 1-A4,5-A11 and 5-E11.,respectively.The titers of ascites prepared by three hybridoma cells were 1:1.02 x 106,1:1.02 x 106 and 1:2.04 x 106.The heavy chain of 1-A4 and 5-A11 ascitic monoclonal antibody were both of Ig G1,5-E11 heavy chain was Ig G2 b,and the light chains of three cell ascites were all of kappa chain.The competitive ELISA test results showed that the monoclonal antibodies produced by three cells were all capable of anti-GTX1～4-KLH.The specific reaction of the envelope was detected,in which the 5-E11 Mc Ab IC50 was least 6.25 micron g/m L.The cross reaction.results showed that there was no cross reaction between the 5-E11 monoclonal antibody and the common other marine toxins.When the concentration of sodium citrate was 5% and the reaction time was 10 min,and the colloidal gold solution with larger size and lower dispersion particle could be obtained.The best coupling effect of nanogold and ascitesumab can be achieved.achieve under the conditions of coupling time set at20 min and antibody solution diluted 5 times.The nitrocellulose membrane needs needs to bedried at 24℃ for 24 h in order to reach a stable state.The stability test results show the basic properties of the test strip are unchanged within three months at room temperature.Based on this,the method of colloidal gold immunochromatographic test strips for the toxin GTX1-4 was preliminarily established,and the lowest detectable limit was up to 12.5μg/mL.更多还原