【作者】 王艺熹；

【导师】 郑晓冬；

【作者基本信息】 浙江大学 ， 食品安全， 2008， 硕士

【摘要】 沙丁胺醇（Salbutamol,Sal）是人工合成的水杨醇类β-肾上腺素激动剂,化学名为1-（4-羟基-3羟甲基苯基）-2-（叔丁胺基）乙醇硫酸盐,是一种选择性β2-受体激动剂。临床上Sal主要用于支气管哮喘的治疗。近年来,因发现Sal具有促进肌肉沉积的作用,可用作牛、羊、禽、猫等畜禽的促生长剂。Sal促生长作用与克伦特罗（Clenbuterol,Clb）类似,而且体内代谢时间又较Clb短暂、毒副作用也相对较弱,因此,目前Sal已成为继Clb之后营养重分配剂的首选药物。但Sal在食品动物中的残留会导致严重的食品安全性问题,我国农业部明文规定禁止将Sal在动物食品中使用,然而受经济利益的驱使,Sal非法使用事件时有发生。因此,建立一种灵敏度高、特异性强、操作简便、适用于大批量样品筛选的Sal检测方法是一项非常紧迫的任务。本研究对Sal人工抗原的合成、动物免疫、Sal兔单抗制备及ELISA检测方法建立等进行了研究,主要结果如下:1、本试验首先将Sal小分子改造成琥珀酸衍生物（Sal-HS）,然后采用混合酸酐法成功合成了两种人工抗原:免疫抗原Sal-BSA和包被抗原Sal-OVA。通过SDS-PAGE电泳证实Sal与BSA、OVA成功偶联,经考马斯亮蓝法测得Sal-BSA的蛋白质浓度为0.7mg/ml。2、将合成成功的Sal-BSA人工抗原免疫4只健康的NZW兔子,2个半月后获得相应的高效价多抗血清。根据ELISA试验结果,最后筛选出A1091编号的兔子血清作为后续试验备用。3、取得兔子脾脏后,采用台盼蓝染色计数法测得脾细胞存活率为96.3%,将脾细胞与处于对数生长期的兔骨髓瘤细胞240E进行细胞融合,其融合率为60%～65%。细胞融合后三周,用间接ELISA法检测40块96孔细胞培养板中的上清,共获得56个阳性克隆孔,将克隆转移至24孔细胞培养板中进行培养。4、一周后,用间接ELISA测56个克隆的细胞培养上清,共获得阳性孔43个,其中双阳性孔31个。综合比较间接ELISA、IgG定量的结果及细胞生长状态,选择Huam-003-6、Huam-003-11、Huam-003-16、Huam-003-22、Huam-003-29五个多克隆进入亚克隆,同时选择Huam-003-18、Huam-003-23、Huam-003-24、Huam-003-35、Huam-003-42多克隆进行冻存处理。5、将亚克隆的阳性孔转移至24孔板培养,共获得经过克隆化的细胞株8株,采用间接ELISA、间接竞争ELISA法筛选,最终筛选出Huam-6-1、Huam-6-4、Huam-6-8、Huam-11.3、Huam-11-8、Huam-16-3、Huam-16-8共7株高特异性的杂交瘤细胞进行冻存,并选取Huam-11-8进行生产。6、从Huam-11-8杂交瘤细胞分离mRNA,分别克隆其重链和轻链基因,基因经酶切后与质粒连接,涂板挑取克隆,大肠杆菌过夜培养提取质粒,重链轻链质粒配对转染至293T细胞进行表达,无菌收集细胞,离心后取培养上清。本试验分两次转染,第一次小量转染后获得了6株Sal单抗质粒。通过间接ELISA试验和间接竞争ELISA试验确定进入第二次大规模转染的Sal单抗质粒为Huam003-11-8-H2/L2。7、通过重组抗体技术生产Sal单抗,采用ProteinA纯化方法纯化并收集Sal单抗,共得到9mlSal单抗。8、对重组兔Sal单抗ELISA检测条件进行了优化,确定以下条件为ELISA最佳条件:Sal-OVA经500倍稀释,37℃包被2h;选择0.5%明胶作为封闭液,37℃封闭2.5h;一抗的工作效价为20000倍,最佳反应条件为37℃,60min;二抗经5000倍稀释后的最佳反应条件为37℃,80min;对底物配方进行优化,得到TMB和H₂O₂的最佳组合为0.275mg/ml的TMB+0.015%的H₂O₂,显色条件为37℃,10min。9、采用间接竞争ELISA法测定Sal单抗的亲和性,结果显示Sal单抗的IC50浓度为9.01ng/ml,IC10浓度为0.81ng/ml,说明本试验制备得到的Sal单抗具有良好的亲和性。10、采用间接竞争ELISA法测定Sal单抗的特异性。从β-兴奋剂、抗生素、喹诺酮类药物、磺胺类药物、激素、农药等不同种类药物中选取了15种小分子进行交叉反应性测定。结果显示,本试验制得的Sal单抗除了与Clb具有22.47%的交叉反应性以外,与其余物质均没有交叉反应性,说明本试验制备的Sal单抗特异性较理想。11、对ELISA法测定样品的准确度进行试验,结果表明不同浓度的尿样及猪肉样品检测的回收率总体在75%～120%之间;本实验中不同添加浓度的样品变异系数大都在10%以下,说明采用本ELISA检测试剂盒测定尿样和猪肉样品具有较好的准确性。12、本试验采用间接竞争ELISA法分别在PBS缓冲液背景、5倍稀释尿样背景及5倍稀释猪肉样品背景中测得3条标准曲线,分别为:y=38.249x+13.484（R²=0.990,IC10=0.81 ng/mL,IC50=9.01ng/ml,线性区间为0.5～100ng/ml）;y=36.599x+3.970（R²=0.982,IC10=1.46ng/mL,IC50=18.10ng/ml,线性区间为1～100ng/ml）;y=45.371x+2.127（R²=0.992,IC10=1.49ng/mL,IC50=11.35ng/ml,线性区间为1～50ng/ml）;其中x为Sal浓度（ng/ml）对数,y为抑制率。更多还原

【Abstract】 Salbutamol（Sal）,also called tetrahydro-isowuinolin,is a kind ofβ₂-receptor agonist which can selective agitate theβ₂-receptor of bronchial smooth muscle and it is used to treat bronchial asthma clinically.Since Sal can also modify growth and repartition nutrition of animals so it was used as growth-promoter on animals.Accumulation ofβ₂-adrenergic agonist in animals is dangerous for consumers and it is forbidden to useβ₂-adrenergic agonists as growth accelerant in many counties,etc. Span,Holland,France and China.The purpose of this study was to synthesize and identify immunogen Sal-BSA/OVA and prepare the rabbit monoclonal antibody（RMcAb） against Sal in order to establish a convenient,effective and quick ELISA method for Sal residues detection at last.To date,there are no reports on RMcAb for Sal residues detection.In the present study,we synthesized the immunogen of Sal,immunized rabbits and obtained immunized spleen of rabbit. Hybridoma and recombinant antibody technology was used to produce a rabbit monocional antibody against Sal.We established ELISA fast detection process and ELISA kit for Sal.The results of this study were reported below:1.Sal was coupled to carrier protein with BSA and OVA by succinic anhydride and cardodiimide. Then 72h dialysis was required to purify the compounds.The final compounds were identified by SDS-Page Electrophoresis.Bradford protein assay was used to detect the final concentrations of SaI-BSA and the protein concentration of Sal-BSA was 0.7mg/mL.2.Four NZW rabbits with serial number A-1090,A-1091,A-1092 and A-1093 were immunized by Sal-BSA.The subcutaneous and venous injection methods were used.Anti-sera with high titers were obtained after six times immunization.3.After immunization,immunized rabbit spleen was obtained.Splenocytes from rabbit were fused with 240E cells using 50%PEG.Fusion cells were plated into a total of 40 96-well culture plates.HAT selection medium was used.Indirect ELISA,indirect competitive ELISA,and IgG quantitative assay were used for screening clones.56 positive clones were selected by primary screening.Huam-003.6-1, Huam-003.6-4,Huam-003.6-8,Huam-003.11-8,Huam-003.11.3,Huam-003.16-3 and Huam-003.16-8 clones were selected after the first subclone.After using indirect ELISA and indirect competitive ELISA, Huam-003.11-8 was selected for the first transfection.4.RMcA-Sal was produced by recombinant antibody.After the first transfection,six strains of RMcA-Sal were obtained.Indirect ELISA and competitive indirect ELISA were used to determine which strain of RMcA-Sal should be taken into the second transfection in order to obtain plenty of RMcA-Sal for the further research.The results showed that Huaan-003.11-8-H2/L2 was the best choice.5.The screening process of ELISA was optimized and the best reaction conditions were selected as follows:96 ELISA plate coated by Sal-OVA with 500-fold diluted was incubated at 37℃for 2h,the primary antibody with titer of 20000-fold was incubated at 37℃for 60min,the second antibody with titer of 5000-fold was incubated at 37℃for 80min,and color was developed and incubated at 37℃for 10min.TMB and H₂O₂ were essential to the sensitivity of ELISA process.The best used concentrations of TMB and H₂O₂ were 0.275mg/ml TMB and 0.015%H₂O₂ respectively.6.The standard curve of Sal-McAb was established by inhibition indirect ELISA.The linear range of the inhibition rate vs log Sal competition curve generated in PBS was 0.5～100ng/mL.And the IC₁₀ and IC₅₀ value of Sal were 0.81ng/mL and 9.01ng/mL.It showed that the Sal-McAb had good affinities.7.Indirect competitive ELISA was used to detect the specificity of Sal-McAb.All cross-reactivity rates except clenbuterol（Clb） were less than 0.01%which means compounds had no cross-reactivity to Sal-McAb except Clb.Cross-reactivity rate of Clb was 22.47%.Veterinary drugs in common use such as chloramphenicol,ampicillin, penicillin,acheomycin,sulfadiazine,enrofloxacin etc.had no cross-reactivity with Sal-McAb.These results showed that specificity of Sal-McAb was high.8.Veracity experiments of indirect ELISA assay showed that the recovery rates of urine and pork samples with different concentration were between 75%～120%.Coefficient variances were generally below 10%.These results showed that the ELISA assay had good accuracy.9.Three standard curves were obtained using indirect competitive ELISA in PBS Buffer matrix, 5-fold diluted urine matrix and 5-fold diluted pork matrix individually:y=38.249x+13.484, y=36.599x+3.97,y=45.371x+2.1269（x here stands for Sal log10（ng/ml） concentration,y here stands for inhibit rate）.更多还原