甲壳类和贝类原肌球蛋白的广谱单克隆抗体制备与鉴定

原肌球蛋白（Tropomyosin, TM）被认为是甲壳类和贝类的主要过敏原，为了应对与日俱增的甲壳类和贝类过敏风险，本研究以南美白对虾（Litopenaeus vannamei）的TM富集液为抗原免疫BALB/c小鼠，通过杂交瘤细胞融合技术筛选获得1株南美白对虾过敏原TM的单克隆抗体细胞株2F8，并利用ELISA、Western Blot、生物信息学等方法对2F8单抗进行免疫学特性分析，确定其效价、特异性、抗体亚型、结合区域，以TM的特异性单抗2F8和兔多抗构建双抗夹心胶体金试纸条分析单抗2F8的应用可行性。结果显示，单抗2F8的抗体亚型为IgG1型、效价为1.28×105，抗体与TM抗原的IC50值为20.21，亲和力高；纯化的单抗2F8可以特异性识别甲壳类、贝类和鱿鱼的主要过敏原TM，存在3个潜在的TM抗原结合区域AA0-31，AA38-43和AA58-67。制备的试纸条检测结果显示，该试纸条可在10 min内目测5 ng/mL的南美白对虾TM（相当于12.5 μg/kg），且可用于甲壳类和贝类肌肉中TM 的检测，研究表明所制备的2F8单抗亲和力高，可用于虾蟹贝和鱿鱼中的过敏原TM的鉴定与检测，具有较好的应用价值。本研究为过敏原TM的鉴定和检测提供了重要生物材料，也为今后TM的快速检测试剂盒开发提供了技术支持。

Preparation and characterization of universal monoclonal antibody specific for tropomyosin in crustacean and shellfish

Tropomyosin (TM) is considered to be the major allergen of crustaceans and shellfish. To address the increasing risk of crustacean and shellfish allergy, in this study, TM enrichment from shrimp (Litopenaeus Vannamei) was used as the immunogen of BALB/c mice. A monoclonal antibody (Mab) cell line 2F8 against TM allergen of Litopenaeus Vannamei was obtained by hybridoma cell fusion techonology. The immunological property of Mab 2F8 including titer, specificity, antibody isotype and binding region was characterized using ELISA, Western Blot and bioinformatics. Moreover, its application feasibility was discussed using a sandwich colloidal gold test strips constructed by Mab 2F8 and rabbit polyclonal antibody specific for TM. ELISA and Western Blot results showed that the antibody subtype of Mab 2F8 was IgG1, with antibody titer of 1.28×105, and an IC50 value of 20.21 indicating a high affinity to TM. It was found that the purified Mab 2F8 had specific reaction with the TMs in crustaceans, shellfish and squid. Three potential binding regions in amino acid sequence AA0-31, AA38-43 and AA58-67 of TM were found according to bioinformatics analysis techniques. Detection results by test strips showed that 5 ng/mL TM (equivalent to 12.5 μg/kg) of white prawn shrimp could be visually detected within 10 min, showing that the obtained Mab 2F8 could be used for the detection of TM in the muscle of crustaceans and shellfish. Our study indicated that the purified 2F8 Mab had high affinity and good application value, could be used for identification and detection of allergen TM in shrimps, crabs, shellfish and squid. This study provides important biological materials for the identification or detection of allergen TM. It provides technical support for the development of rapid detection kit of TM in the future.