目的制备抗人ARMET(arginine-rich,mutated in early stage of tumors)的单克隆抗体(mAb),并鉴定其特性。方法将pET28a-ARMET转化到大肠杆菌BL21中,然后用IPTG诱导表达,用预装好的Ni-beads柱进行纯化,通过SDS-PAGE电泳及考玛斯亮蓝染色...目的制备抗人ARMET(arginine-rich,mutated in early stage of tumors)的单克隆抗体(mAb),并鉴定其特性。方法将pET28a-ARMET转化到大肠杆菌BL21中,然后用IPTG诱导表达,用预装好的Ni-beads柱进行纯化,通过SDS-PAGE电泳及考玛斯亮蓝染色方法判断融合蛋白的表达量及纯度。将纯化的ARMET免疫雌性BALB/c小鼠后采用淋巴细胞杂交瘤技术,获取分泌抗ARMET杂交瘤细胞株。体内诱生腹水法制备mAb,间接ELISA法测定其效价及抗体亚型,辛酸-硫酸铵沉淀法及亲和层析法纯化mAb。用免疫荧光双标及Western blot法对抗体的特异性进行了鉴定。结果获得了纯度较高、浓度较高的融合蛋白;建立了7株稳定分泌特异性抗ARMET mAb的杂交瘤细胞株,诱生腹水法获得的抗体效价在1×10-5～1×10-7之间,且均有较好的特异性。结论已成功制备出抗ARMET mAb,为进一步用于临床诊断和实验研究奠定了基础。展开更多
The asymmetric hydrolysis of racemic ibuprofen ester is one of the most important methods for chiral separation of ibuprofen. In this work, a catalytic antibody that accelerates the rate of enantioselective hydrolysis...The asymmetric hydrolysis of racemic ibuprofen ester is one of the most important methods for chiral separation of ibuprofen. In this work, a catalytic antibody that accelerates the rate of enantioselective hydrolysis of ibuprofen methyl ester was obtained against an immunogen consisting of tetrahedral phosphonate hapten attached to bovine serum albumin (BSA). The catalytic activity of the catalytic antibody in the water-miscible organic-solvent system composed of a buffer solution and N, N-dimethylformamide (DMF) was studied. With 6% DMF in the buffer solution (containing catalytic antibody 0.25 μmol, 0.2 mol·L-1 phosphate buffer, pH 8) at 37°C for 10 h, a good conversion (48.7%) and high enantiomeric excess (>99%) could be reached. The kinetic analysis of the cata-lytic antibody-catalyzed reaction showed that the hydrolysis in the water-miscible organic-solvent system with DMF in buffer solution followed the Michaelis-Menten kinetics. The catalytic efficiency (Kcat/Km) was enhanced to 151.91 L·mmol-1·min-1, twice as large as that for the buffer solution only.展开更多
Irbesartan is an antihypertensive drug whose concentration in blood is very small so it requires a sensitive method of analysis, selective and valid for analysis. In this study, it is carried out optimization of analy...Irbesartan is an antihypertensive drug whose concentration in blood is very small so it requires a sensitive method of analysis, selective and valid for analysis. In this study, it is carried out optimization of analytical conditions and validation for the analysis of irbesartan in plasma. Chromatography was performed on a C 18 column （250 × 4.6 mm, 5 μm） under isocratic elution with acetonitrile-0.1% formic acid （46：54 v/v）, pH 3.75. Detection was made at excitation 250 nm and emission 370 nm and analyses were run at a flow-rate of 1.0 mL/min at a temperature of 40 ℃. Losartan potassium was used as internal standard. Plasma extraction was done by deproteination with acetonitrile, mixed with vortex for 30 seconds, then centrifuged it at 10,000 rpm for 10 rain. In plasma validation, the recovery was 96.22%, and the lower limit of quantification （LLOQ） in plasma was 2 ng/mL. The method also fulfill the criteria for accuracy and precision intra and inter day by normal values （%Diff） not exceed ± 15%. On the stability study, irbesartan in plasma temperature -20 ℃has been stable for 28 days.展开更多
文摘目的制备抗人ARMET(arginine-rich,mutated in early stage of tumors)的单克隆抗体(mAb),并鉴定其特性。方法将pET28a-ARMET转化到大肠杆菌BL21中,然后用IPTG诱导表达,用预装好的Ni-beads柱进行纯化,通过SDS-PAGE电泳及考玛斯亮蓝染色方法判断融合蛋白的表达量及纯度。将纯化的ARMET免疫雌性BALB/c小鼠后采用淋巴细胞杂交瘤技术,获取分泌抗ARMET杂交瘤细胞株。体内诱生腹水法制备mAb,间接ELISA法测定其效价及抗体亚型,辛酸-硫酸铵沉淀法及亲和层析法纯化mAb。用免疫荧光双标及Western blot法对抗体的特异性进行了鉴定。结果获得了纯度较高、浓度较高的融合蛋白;建立了7株稳定分泌特异性抗ARMET mAb的杂交瘤细胞株,诱生腹水法获得的抗体效价在1×10-5～1×10-7之间,且均有较好的特异性。结论已成功制备出抗ARMET mAb,为进一步用于临床诊断和实验研究奠定了基础。
基金Supported by the Natural Science Foundation of Zhejiang Province (Y404353)
文摘The asymmetric hydrolysis of racemic ibuprofen ester is one of the most important methods for chiral separation of ibuprofen. In this work, a catalytic antibody that accelerates the rate of enantioselective hydrolysis of ibuprofen methyl ester was obtained against an immunogen consisting of tetrahedral phosphonate hapten attached to bovine serum albumin (BSA). The catalytic activity of the catalytic antibody in the water-miscible organic-solvent system composed of a buffer solution and N, N-dimethylformamide (DMF) was studied. With 6% DMF in the buffer solution (containing catalytic antibody 0.25 μmol, 0.2 mol·L-1 phosphate buffer, pH 8) at 37°C for 10 h, a good conversion (48.7%) and high enantiomeric excess (>99%) could be reached. The kinetic analysis of the cata-lytic antibody-catalyzed reaction showed that the hydrolysis in the water-miscible organic-solvent system with DMF in buffer solution followed the Michaelis-Menten kinetics. The catalytic efficiency (Kcat/Km) was enhanced to 151.91 L·mmol-1·min-1, twice as large as that for the buffer solution only.
文摘Irbesartan is an antihypertensive drug whose concentration in blood is very small so it requires a sensitive method of analysis, selective and valid for analysis. In this study, it is carried out optimization of analytical conditions and validation for the analysis of irbesartan in plasma. Chromatography was performed on a C 18 column （250 × 4.6 mm, 5 μm） under isocratic elution with acetonitrile-0.1% formic acid （46：54 v/v）, pH 3.75. Detection was made at excitation 250 nm and emission 370 nm and analyses were run at a flow-rate of 1.0 mL/min at a temperature of 40 ℃. Losartan potassium was used as internal standard. Plasma extraction was done by deproteination with acetonitrile, mixed with vortex for 30 seconds, then centrifuged it at 10,000 rpm for 10 rain. In plasma validation, the recovery was 96.22%, and the lower limit of quantification （LLOQ） in plasma was 2 ng/mL. The method also fulfill the criteria for accuracy and precision intra and inter day by normal values （%Diff） not exceed ± 15%. On the stability study, irbesartan in plasma temperature -20 ℃has been stable for 28 days.
