| 摘要: |
| 【目的】制备无色孔雀石绿（LMG）单克隆抗体，并分析其生物学特性，为建立LMG残留的免疫学检测方法奠定基础。【方法】通过改进的碳二亚胺法制备无色孔雀石绿 牛血清白蛋白偶联物(LMG-BSA)与副品红 牛血清白蛋白偶联物（PA-BSA），用紫外扫描、质谱法方法鉴定偶联物。利用制备的LMG-BSA与PA-BSA作为免疫原，免疫4～5周龄Balb/c小鼠，用杂交瘤技术制备LMG单克隆抗体，并对其效价、亲和力、灵敏度和特异性等进行分析。【结果】LMG-BSA与PA-BSA的偶联比率分别为7∶1和37∶1；经间接酶联免疫吸附（ELISA）法筛选，由LMG-BSA和PA-BSA抗原各获得1株杂交瘤细胞，分别命名为L-1、P-1。这2株细胞株分泌的单克隆抗体纯化后，经测定效价分别为1.25×105和6.25×106，亲和力常数分别为4.92×109和1.44×108 L/mol，半抑制质量浓度（IC50）分别达8.03和10.5 μg/L。L-1分泌的单克隆抗体仅与孔雀石绿存在交叉反应，交叉反应率为100%；P-1分泌的单克隆抗体与孔雀石绿及副品红均存在交叉反应，交叉反应率分别为80%与100%。【结论】成功获得了2株LMG单克隆抗体，其均可用于水产品中LMG残留的快速检测。 |
| 关键词: 无色孔雀石绿 副品红 半抗原 抗体 免疫 |
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| 基金项目:现代农业产业技术体系建设专项资金项目（NYCYTX-49-17）；公益性行业（农业）科研专项经费项目（nyhyzx07-042）；公益性行业（农业）科研专项经费项目（200803013） |
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| Preparation of two different monoclonal antibodies against leuco malachite green |
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| Abstract: |
| 【Objective】The research was conducted to generate monoclonal antibodies of leuco malachite green (LMG) and analyze its biological traits to establish a determination method for LMG residues in food tissues.【Methods】The conjugation of leuco malachite green bovine serum albumin (LMG-BSA) and paraleucaniline-bovine serum albumin (PA-BSA) were prepared via a modified carbodiimide method.Features of the conjugation were identified by ultraviolet spectrometry and mass spectroscopy.In order to generate monoclonal antibody (Mab) against hapten-leuco malachite green (LMG),LMG-BSA and PA-BSA were used as immunogen to immunize for four to five-week-old Balb/c mice.The Mabs were obtained by hybridoma technology and their titer,affinit,sensibility and specificity were identified.【Results】LMG-BSA and PA-BSA coupling ratio was 7∶1 and 37∶1;The titers of purified Mabs secreted by L-1 and P-1 were 1.25×105,6.25×106,respectively.Characterization studies of these two Mabs showed that they had high affinity for LMG,with the affinity of 4.92×109,1.44×108 L/mol,and the IC50 was 8.03 and 10.5 μg/L.Mabs secreted by L-1 exhibited high cross reactivity(100%) with malachite green;Mabs secreted by P-1 exhibited high cross-reactivity(80% and100%) with malachite green and paraleucaniline.【Conclusion】These two monoclonal antibody were useful for determination of LMG residues in aqua-production. |
| Key words: leuco malachite the green paraleucaniline hapten antibody immunity |
