一种新的沙丁胺醇人工抗原的合成及单克隆抗体的制备
Synthesis of a newly designed artificial antigen and preparation of a monoclonal antibody against salbutamol
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摘要: 【目的】为证明采用 4 - 溴丁酸乙酯半抗原合成人工抗原可制备灵敏度高、特异性好的单克隆抗体.【方法】将硫酸沙丁胺醇（SAL）与 4 - 溴丁酸乙酯反应制备半抗原（SAL-HS）；通过 N - 羟基琥珀酰亚胺活化酯法合成免疫原与包被原，用聚丙烯酰胺凝胶电泳、紫外光谱和质谱方法对沙丁胺醇免疫原及包被原进行鉴定.免疫原免疫 Balb/c 小鼠，采用融合技术产生杂交瘤细胞株，体内诱生腹水法制备单克隆抗体，并鉴定其免疫学特性.【结果和结论】筛选出1株杂交瘤细胞株，其细胞培养上清液效价达1∶128 000，腹水效价达1∶2 560 000，免疫球蛋白亚型鉴定为 IGg1；抗体对沙丁胺醇的半数抑制浓度（IC50）为：0.746 ng/mL；与其他同类结构药物的交叉反应率均小于0.015%.Abstract: 【Objective】 To prove that the artificial antigen which was synthesised by 4-bromine ethyl butyrate was successful，and that the monoclonal antibody had a high sensitivity and specificity.【Method】 Salbutamol sulfate was connect with 4-bromine ethyl butyrate，then converted into hapten（SAL-HS）.The immunogen SAL-BSA(salbutamol-bovine serum albumin) and coating antigen SAL-OVA（ovalbumin）were synthesized by the N-Hydroxy-succinimide active ester method. The conjugations were identified by SDS-PAGE,UV and mass spectrum.Balb/c mice were immunized by immunogen; splenocytes were fused with SP2/0 myeloma cells; hybridomas cells were produced for a monoclonal antibody by ascites in vivo to identify immunological characteristics.【Result and conclusion】 Cultural supernatants titer of hybridomas cells and ascites were 1∶128 000 and 1∶2 560 000 respectively; the subclasses of monoclonal antibody was IGg1; the IC50 of standard curve was 0.746 ng/mL, with other drugs of similar structure showing cross reactivity affinities being less than 0.015%.
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Keywords:
- salbutamol /
- monoclonal antibody /
- immunogen /
- ic-ELISA
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