水产品中两种土腥味物（土嗅素、二甲基异茨醇）单克隆抗体的制备及其高灵敏快速检测方法的建立

Aquatic organisms like fish can easily absorb the earthy-musty odorants from water body, making the muscle of the aquatic organisms smelly, which decreases their taste and edible value and leads to a great economic loss for aquaculture. Geosmin (GSM) and 2-methylisoborneol (MIB) are two major earthy-musty odorants, which are the metabolites of filamentous bacteria (actinomycetes) and blue green algae (cyanobacteria) overgrowing in the eutrophicated water body. The threshold values of the GSM and MIB in water body and fish body are very low. At present there is no report of the high sensitive and fast analytical method for the detection of GSM and MIB. In this proposal, GSM and MIB are selected as the target analytes. According to the principle of hapten molecular design and the characterization of molecular structure of GSM and MIB, we have designed and will synthesize the modified derivatives which utmost maintain the whole molecular structure of the parent target analyte. Then we will link the modified derivative to carrier protein to form hapten-protein conjugate which will be used as the immunogen for the antibody (Ab) production. Taking advantage of hybridoma technology, we will respectively produce monoclonal antibodies (mAb) against GSM and MIB. Based on the prepared mAb, we will combine the ultrasensitive surface-enhanced Raman spectroscopy (SERS) with the convenient immunochromatographic assay (ICA) to establish a highly sensitive and specific, simple and fast analytical method named as SERS-ICA for the determination of GSM and MIB in aquatic products. We will apply the developed SERS-ICA for real sample analysis. This proposal displays a strong creation and wide application, and exhibits a big scientific significance and application value.

水生生物极易吸附水体中的土腥味物，使肌肉中产生难闻气味，影响食用口感，给水产养殖业造成巨大的经济损失。土嗅素(GSM)和二甲基异茨醇(MIB)是两种最主要的土腥味物，为富营养化水体中过度生长的放线菌、蓝绿藻的代谢物; GSM和MIB在水体和鱼体中的嗅阈值很低，目前尚无高灵敏快速检测这两种物质的文献报道。我们拟以GMS及MIB为目标物，根据半抗原分子设计理论和目标物分子结构特点，设计并合成保持目标物母体较完整分子结构的修饰物，将修饰物与载体蛋白相连制得免疫原，运用杂交瘤技术制备抗GSM和MIB的单克隆抗体(mAb)，将灵敏度高的表面增强拉曼光谱(SERS)及便捷的免疫层析技术(ICA)相结合，建立灵敏度高、特异性强、简便、快速的检测水产品中GMS及MIB的表面增强拉曼光谱免疫层析分析方法(SERS-ICA)，并将其应用于实际样品的检测。本项目创新性强，适用性广，具有较大的科学意义和应用价值。

水生生物极易吸附水体中的土腥味物，使肌肉中产生难闻气味，影响食用口感，给水产养殖业造成巨大的经济损失。二甲基异茨醇(MIB)和土嗅素(GSM)是两种最主要的土腥味物，为富营养化水体中过度生长的放线菌、蓝绿藻的代谢物; GSM和MIB在水体和鱼体中的嗅阈值为ppt级，目前尚无高灵敏快速检测这两种物质的文献报道。氯霉素及其替代物氟苯尼考是一类水产养殖中广泛使用的抗生素，抗菌性强，毒副作用大，在鱼饲料中加入过量，将使水产品中残留量过高，对消费者的健康造成较大损害，故必须对水产品中这类物质进行高灵敏快速检测。根据MIB、GSM、氯霉素和氟苯尼考的分子结构特点，分别合成了保持较完整原有分子架构的四种修饰物，并与载体蛋白相联，制得免疫原和包被抗原，运用杂交瘤技术，制得抗MIB、GSM、氯霉素和氟苯尼考的四种单抗。由于抗MIB和GSM的两种单抗的灵敏度达不到ppt级，故将它们分别共价交联于琼脂糖凝胶上并填充于空的SPE柱中，用制备出的免疫亲合柱分离萃取样品中MIB和GSM，并与GC-MS结合，建立了基于免疫亲合柱的检测MIB和GSM的GC-MS方法，检出限均为2 ppt，能满足实际样品的检测要求。抗氯霉素和抗氟苯尼考单抗的灵敏度分别1.6 ppb和2.0 ppb，利用这两种单抗及本实验室所制备的其它抗体，分别建立了检测氯霉素、氟苯尼考及其它有毒有害物的多种免疫分析方法如ELISA、电化学免疫传感器、化学发光免疫分析法、免疫层析分析方法等，成果丰硕，有些具有产业化前景。制备了多种具有较大SERS增强效果的贵金属纳米颗粒如Au纳米棒、Au @Ag纳米棒、Ag@Au纳米颗粒、Au三角片纳米颗粒、SiO2包裹的Ag纳米颗粒等，广泛深入开展了表面增强拉曼光谱免疫层析分析方法(SERS-ICA)的研究，取得了较满意的成果，己与多家仪器公司商谈，希望共同开发出高灵敏、高特异的SERS-ICA读卡仪。