细交链孢菌酮酸（Tenuazonic acid，TeA）毒性位于链格孢霉毒素之首，具有致畸、致癌等多种毒副作用。近年来在谷物、果蔬等各类农产品及食品中均有检出，严重危害到人民群众身体健康，因此加强食品中TeA的检测十分重要。仪器分析法具有灵敏度高、准确性好等优点，但无法满足现代食品安全对于快速、简便、低成本等要求。免疫分析方法因具有灵敏、快速、高通量等特点可满足食品安全快速筛查需求，已发展成为TeA检测领域的研究热点。本文以TeA为研究对象，目标在于制备TeA单克隆抗体并建立两种免疫分析方法用于TeA的检测，主要研究内容如下：

1. TeA半抗原及完全抗原的合成及表征

本文总结出5种TeA半抗原，结合分子模拟技术选择并合成3种：TeA、半抗原H1和半抗原H2，其中半抗原H1和H2由TeA衍生化制备。通过液相质谱、核磁共振氢谱、紫外光谱及红外光谱鉴定这3中半抗原。采用不同方法与载体蛋白（牛血清白蛋白、卵清蛋白）偶联制备四种免疫原和四种包被原。通过紫外光谱、SDS-PAGE对人工抗原进行鉴定并测定其蛋白浓度。

2. TeA单克隆抗体的制备及ic-ELISA方法的建立

使用上述四种免疫原免疫小鼠，经过细胞融合、杂交瘤细胞筛选及亚克隆筛选得到3C7和4F7两株稳定分泌抗体的杂交瘤细胞株，选择4F7制备腹水并纯化得到抗TeA单克隆抗体。基于单克隆抗体建立ic-ELISA检测方法，ic-ELISA方法最佳反应条件为包被原TeA-OVA-CDI浓度0.36 µg/mL，抗体浓度0.45 µg/mL，TeA标准品储备液用0.01 M PBS（pH 7.4，含0.2% NaCl）梯度稀释，竞争反应时间40 min，二抗反应时间30 min，显色时间15 min。在最优条件下ic-ELISA方法的IC₅₀为20.50 ng/mL，线性检测范围（IC₂₀~IC₈₀）为3.30~165.00 ng/mL，检测限（IC₁₀）为1.20 ng/mL。与AOH、AME、AFB1、OTA不存在交叉反应。番茄、葡萄、枸杞和红枣样品中加标回收率在88.78~117.53%之间。

3. TeA胶体金免疫层析试纸条的制备

利用柠檬酸钠还原法制备胶体金纳米粒子并偶联抗体制备金标抗体探针，对胶体金免疫层析试纸条的实验条件进行了优化，其最优条件为：金标探针0.1 M K₂CO₃添加量为2 µL每毫升胶体金，抗体添加量为6 µg每毫升胶体金，金标探针稀释倍数为3×，T线包被原浓度为0.25 mg/mL。在最优条件下对试纸条进行标准样品检测，利用ImagJ分析得到标准曲线线性方程为：y=5665.06-9.89x，R²=0.98。线性检测范围（IC₂₀~IC₈₀）为105.18~455.90 ng/mL，检测限（IC₁₀）为46.73 ng/mL，肉眼观察试纸条的消线值为500 ng/mL。试纸条特异性良好，与AOH、AME、AFB1、OTA无交叉反应。番茄、葡萄、枸杞和红枣实际样品加标回收率在90.60%~100.23%之间。

Tenuazonic acid (TeA) has many side effects such as carcinogenesis, which toxicity is the first toxin of Alternaria mycotoxin. Recently, it has been detected in various agricultural products and foods such as grains, fruits and vegetables, seriously endangering people's health. Therefore, it is important to strengthen the detection method of TeA in food. Instrumental analysis method has the advantages of high sensitivity and good accuracy, but it can not meet the requirements of fast, simple and low cost for modern food safety. Immunoassay has become a research hots pot in the field of TeA detection because of its sensitivity, rapidity and high throughput, which can meet the needs of rapid food safety screening. This thesis takes TeA as the research object, aiming to prepare TeA monoclonal antibody and establish two immunoassay methods for the detection of TeA. The main research contents are as follows:

1. Synthesis and identification of TeA hapten and artificial antigen

In this study, five kinds of TeA haptens were summarized, and the single point energy, optimal spatial conformation and molecular orbital of each hapten were calculated by molecular simulation technology. TeA, hapten 1 and hapten 2 were selected and synthesized, among which hapten 1 and hapten 2 were prepared by TeA molecular derivatization. Four kinds of immunogen and four kinds of coated antigen were prepared by coupling with carrier proteins (bovine serum albumin and ooalbumin) by different methods. The artificial antigens were identified by UV and SDS-PAGE.

2. TeA monoclonal antibody was prepared and ic-ELISA assay was established

After cell fusion, hybridoma cell screening and subclonal screening, two hybridoma cell lines 3C7 and 4F7 with stable antibody secretion were obtained. 4F7 was selected to prepare ascites and purify monoclonal antibody of TeA. An ic-ELISA was established based on monoclonal antibody for TeA. The optimal reaction conditions for ic-ELISA were as follows: concentration of TeA-OVA-CDI was 0.36 µg/mL, concentration of monoclonal antibody was 0.45 µg/mL, diluent of TeA stock solution was 0.01 M PBS (pH 7.4, 0.2% NaCl). The reaction time of competitive response was 40 min, The secondary reaction with the enzyme-labeled conjugate was allowed durinng 30 min, and the color developing reaction at 15 min. Under the conditions above, The IC₅₀ value for TeA was estimated to be 20.50 ng/mL with the linear detection range (IC₂₀-IC₈₀) of 3.30 to 165.00 ng/mL, and the limit detection of the assay was 1.20 ng/mL. There was no crossing reaction with AOH, AME, AFB1 and OTA. The recoveries of tomato, grape, wolfberry and jujube were 88.78% to 117.53%.

3. Establishment of a colloidal gold immunochromatographic strip for TeA detection

Colloidal gold nanoparticles were prepared by sodium citrate reduction method and gold-labeled antibody probes were prepared. The experimental conditions of colloidal gold immunochromatographic strips were optimized. The optimal conditions were as follows: The pH of the gold-labeled probe was 2 µL 0.1 M K₂CO₃ per mL colloidal gold, the antibody addition was 6 µg antibody per mL colloidal gold, the dilution ratio of the gold-labeled probe was 3× and the concentration of T-line coating antigen was 0.25 mg/mL. The performance of the strip detection method was evaluated under optimal conditions, and the standard curve obtained by ImagJ analysis was y=5665.06-9.89x (R²=0.98). The linear detection range (IC₂₀~IC₈₀) was 105.18 to 455.90 ng/mL, the limit of detection (IC₁₀) was 46.73 ng/mL, and the test strip had good specificity and no cross-reaction with AOH, AME, AFB1 and OTA. The recoveries of tomato, grape, wolfberry and jujube were 90.60% to 100.23%.

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