蓝藻水华频繁暴发，水体中蓝藻毒素的监测具有重要意义。蓝藻毒素具有多种结构变异体，发展基于广谱性抗体的免疫分析技术可实现对一类蓝藻毒素的快速高灵敏分析，对满足蓝藻毒素快速低成本筛查和预警监测需求具有重要的科学意义与现实价值。本研究以蓝藻毒素中的微囊藻毒素（MCs）和节球藻毒素（NODs）为研究对象，提出新型复合免疫原策略制备MCs和NODs（MCs/NODs）广谱特异性单克隆抗体，揭示抗体广谱识别机制；在此基础上建立总MCs/NODs快速检测的酶联免疫吸附测定（ELISA）方法，开展环境应用研究并研发标准化检测流程；引入基于纳米酶正交反应的信号放大，研发MCs/NODs快速检测的便携式纸芯片。主要研究结果：（1）提出新型复合免疫原设计方案，基于该设计成功制备了针对MCs/NODs的广谱特异性单克隆抗体。获得5株分泌IgG1型抗体（轻链均为κ型）的阳性杂交瘤细胞株。制备的5株IgG1型抗体均可特异性识别13种MCs/NOD结构变异体，具有广谱特异性，且纯度、浓度和活性均可满足后续的免疫检测要求。（2）构建了抗体的三维结构，初步揭示了抗体的广谱识别机制，并建立了检测水中总MCs/NODs的间接竞争ELISA方法。5株抗体对12种MCs结构变异体均具有较高的亲和力，平均亲和常数分别为4.10×108、1.12×109、5.35×108、6.64×108与3.92×108 L/mol。Adda为抗体识别MCs/NODs的共有抗原表位，导致了抗体对含有Adda的配体分子具有广谱识别性。ELISA方法对13种MCs/NODs结构变异体的检出限均低于0.3 μg/L，交叉反应性为60~127%。（3）建立了针对饮用水体和环境水体的间接竞争ELISA方法标准化检测流程。在降低水体基质效应基础上，加标回收率在80~117%之间。基于我国与美国分别推荐的样品预处理方式，在所测试的地表水体中，后者测得的MCs/NODs浓度是前者的1~5倍；基于美国预处理方式，在几类地表水体中测得的总MCs/NODs浓度均低于美国环境保护署对学龄儿童和成人饮用水中所推荐的限值。（4）基于智能手机集成了便携化纸芯片光学成像系统，提出了逐层生物正交点击反应介导纳米酶催化的信号放大策略，建立了检测总MCs/NODs的纸芯片ELISA方法。成像系统可实现对4通道阵列式纸芯片的同步检测。纸芯片ELISA方法对13种MCs/NODs结构变异体的检出限均低于0.7 μg/L，交叉反应性为38~105%，加标回收率在87~120%之间，具备对环境水体中MCs/NODs的广谱分析能力。

It is important to monitor cyanotoxins in water since the blue-green algae blooms occur frequently. Cyanotoxins exist in form of many structural variants. The broad-spectrum antibodies-based immunoassay techniques can realize rapid and sensitive quantitative detection of a class of cyanotoxins in water, providing important scientific significance and practical value to meet the needs of rapid and low-cost screening and early warning monitoring of cyanotoxins. In this study, microcystins (MCs) and nodularins (NODs) were took as research objects. A novel multi-immunogen strategy was proposed for the production of broad-spectrum monoclonal antibodies against MCs and NODs (MCs/NODs), as well as the broad-spectrum recognition mechanism of antibodies was revealed. On this basis, an enzyme-linked immunosorbent assay (ELISA) method for rapid detection of total MCs/NODs in water was established, researches on environmental application were carried out, and a standardized detection process was developed. Besides, a portable paper chip-based immunoassay technique was developed by introducing signal amplification based on bioorthogonal click reaction of nanozymes for rapid detection of MCs/NODs in water.Major results and conclusions obtained in this study are listed as follows: (1) Based on the designed novel scheme for multi-immunogen, broad-spectrum monoclonal antibodies against MCs/NODs were successfully prepared. Five strains of positive hybridoma cell lines that can produce IgG1 antibodies with kappa (κ) light chains were obtained. All the five strains of IgG1 antibodies exhibited broad-spectrum specificity and can specifically recognize thirteen MCs/NODs variants, besides, their purity, concentration and activity can meet the requirements of the follow-up immunoassay. (2) The 3d structure of antibodies was constructed, the broad-spectrum recognition mechanism of antibodies was revealed, and an indirect competitive ELISA method was established for the detection of MCs/NODs in water. All the five strains of antibodies exhibited quite high and uniform affinities against twelve MCs variants with the average binding affinity constants calculated to be 4.10×108, 1.12×109, 5.35×108, 6.64×108, and 3.92×108 L/mol respectively. The Adda moiety may be the common epitope for the recognition of MCs/NODs by antibodies, leading to broad-spectrum recognition against Adda-containing ligand molecules by antibodies. Thirteen MCs/NODs variants were detected by using the established ELISA method, and all the limits of detection (LODs) were below 0.3 μg/L, well with the good molar cross-reactivities of 60~127%. (3) The standardized detection process of the indirect competitive ELISA method for drinking water and environmental water was established. On the basis of reducing the matrix effect of water samples, the recovery percentages of spiked ones were 80~117%. In terms of the surface water samples being tested, the concentrations of MCs/NODs measured based on pretreatment method recommended by the United States were 1~5 times that of those measured based on pretreatment method recommended by China. Besides, all the measured total MCs/NODs of several surface water samples based on pretreatment method recommended by the United States were below the health advisory recommendation limit for MCs in drinking water proposed by the United States Environmental Protection Agency for school-age children through adults. (4) Combined with the smartphone, a portable optical imaging system for paper-based chip was integrated, and an ELISA method applied to paper-based chips for MCs/NODs detection was established by designing a signal amplification strategy based on the layer-by-layer bioorthogonal click reaction-mediated nanozyme catalysis. The imaging system meeting the parallel detection requirements for paper-based chips in the form of four-channel arrays. The LODs towards thirteen MCs/NODs variants were below 0.7 μg/L with the molar cross-reactivities were 38~105%. The recovery percentages of spiked water samples were 87~120%, indicating the broad-spectrum analysis ability for MCs/NODs in environmental water.