基于荧光纳米颗粒标记的盐酸克伦特罗荧光免疫层析试纸的研究
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摘要
成功制备了CL免疫原和包被原，免疫小鼠后进行了细胞融合，应用杂交瘤技术筛选出了3株敏感特异的杂交瘤细胞，其中1F8细胞株的细胞培养上清的效价达到了1:1280。用1F8细胞株接种用液体石蜡处理过的小鼠，采集腹水，检测腹水的效价为1:512000，间接竞争ELISA结果显示腹水IC50为2.45ng/mL。CL-McAb的亲和常数（Ka）为4.78×1010L/mol，与其他药物的交叉反应率均小于0.08%，获得了高效价、高敏感性、高亲和力和高特异性的CL-McAb。
采用反相微乳法制备了SiO2纳米颗粒，通过改变DDW的量得到了粒径在14～97nm之间的分散性较好、粒径均一的球形SiO2纳米颗粒。同时采用St ber法制备了SiO2纳米颗粒，通过改变TEOS的量得到了粒径在93～398nm之间的单分散、粒径均一的球形SiO2纳米颗粒。用硅烷偶联剂APTS对制备的SiO2纳米颗粒进行有水条件下的表面修饰，用傅里叶红外光谱仪检测改性结果，证明改性成功。
选择具有优良发光性能的稀土配合物BHHCT-Eu3+为荧光材料，在反相微乳法合成SiO2的基础上，制备了共价型稀土荧光纳米颗粒，通过多次包壳，增加了荧光纳米颗粒的荧光强度。在TEM下，稀土荧光纳米颗粒呈现均匀的球状结构，粒径为59±5nm，具有较好的分散性。利用氧化后Dextran-500k的醛基成功的将带氨基的稀土荧光纳米颗粒和CL-McAb联接，将稀土荧光抗体喷涂在结合垫上，分别选择CL-BSA和SPA作为NC膜上的T线和C线印迹，将样品垫、结合垫、NC膜、吸水纸按照一定的工艺固定在支持板上，从而制成了稀土荧光检测试纸。对稀土荧光检测试纸的性能进行了鉴定，其肉眼观测完全抑制限为1.6ng/mL，通过微型荧光读条仪可以进行定量检测，定量检测范围为0.04～1.27ng/mL，LOD为0.04ng/mL，较同抗体制备的胶体金试纸灵敏许多，具有较好的特异性，与同系物几乎无交叉，具有良好的重复性、准确性，有效期至少为6个月。
选择具有优良发光性能的FITC为荧光材料，利用St ber法制备了掺杂型的FITC-SiO2纳米颗粒，该荧光纳米颗粒具有FITC的荧光特征峰，有较好的耐光性，不易发生荧光泄露。在TEM下，FITC-SiO2纳米颗粒呈现均匀的球状结构，粒径为96±6nm，具有较好的分散性。利用氧化后Dextran-500k的醛基将带氨基的FITC-SiO2纳米颗粒和CL-McAb联接，将FITC荧光抗体喷涂在结合垫上，分别选择CL-BSA和SPA作为NC膜上的T线和C线印迹，将样品垫、结合垫、NC膜、吸水纸按照一定的工艺固定在支持板上，从而制成了FITC荧光检测试纸。对FITC荧光检测试纸的性能进行了鉴定，其肉眼观测完全抑制限为6ng/mL，通过微型荧光读条仪可进行定量检测，定量检测范围为0.16～5.18ng/mL，LOD为0.16ng/mL，较同抗体制备的胶体金试纸灵敏，具有较好的特异性，与同系物几乎无交叉，具有良好的重复性、准确性，有效期至少为6个月。
建立了检测猪尿样品中CL残留的GC-MS方法，其线性检测范围为0.10～500ng/mL，LOD为0.1ng/mL，该法灵敏、准确、稳定，需要昂贵的仪器设备，专门的操作人员，检测周期长，费用较高，无法做到现场高通量检测。比较分析了GC-MS、稀土荧光试纸和FITC荧光试纸对实际样品的测定结果，稀土荧光检测试纸与确证方法GC-MS的符合率为100%，FITC荧光检测试纸与确证方法GC-MS的符合率为97.6%，进一步确证了稀土荧光试纸和FITC荧光试纸检测CL残留具有较好的准确性、时效性。
Two complete antigens of CL-BSA and CL-OVA were successfully prepared.BALB/c mice were immunized with CL-BSA, monoclonal antibody against CL(CL-McAb) was prepared by hybridoma technology. Three hybridoma cell lines werescreened. The hybridoma cell line1F8which the titer was1:1280in supernatant wereinoculated with liquid paraffin treated mice. CL-McAb ascites were collected and tested.The titer of hybridoma cell line1F8was1:512000of in ascites and the indirectcompetitive ELISAresults showed the half inhibitory concentration (IC50) of2.45ng/mLto CL. The affinity constant (Ka) of the CL-McAb was3.75×1010L/mol and thecross-reactivity with other drugs were less than0.08%. CL-McAb with high-titer, highsensitivity high affinity and high specificity had been produced successfully in thisexperiment.
Spherical silica nanoparticles with good dispersion and uniform particle sizebetween14and97nm were successfully abtained by varying the amount of water in thepreparation through inverse microemulsion method. Spherical silica nanoparticles withgood dispersion and uniform particle size between93and398nm were successfullyabtained by varying the amount of TEOS in the preparation through St ber method. Asilane coupling agent APTS was used to modify the surface of the silica nanoparticlesunder the conditions of in water. The Fourier transform infrared spectroscopy resultsprove that the silica nanoparticles have been successful modification.
Rare earth chelates BHHCT-Eu3+with excellent luminescent properties had beenselected to prepare the covalent type rare earth fluorescent nanoparticles based on thesilica nanoparticles by the reverse microemulsion method. The fluorescence intensity ofthe rare earth fluorescent nanoparticles increased through multiple cladding, and thefluorescent nanoparticles appeard good dispersion and uniform particle size of59±5nmwith homogeneous globular structures under TEM. The rare earth fluorescentnanoparticles with amino group had successfully couplied with CL-McAb through thealdehyde groups of the oxidized Dextran-500k. The rare earth fluorescent antibody hasbenn sprayed on the conjugate pad. CL-BSAand SPAwere selected as T line and C lineblot of the NC membrane. The sample pad, conjugate pad, NC membrane and theabsorbent paper were fixed on the support plate according to a certain process toproduce the rare earth fluorescence test strip. The performance of the rare earthfluorescent test strip had been identified. The limit of completely suppressed of the stripwith naked-eye is1.6ng/mL, meanwhile quantitative detection range of test strip usingthe micro-fluorescence analyzer is0.04~1.27ng/mL with the LOD0.04ng/mL that ismuch better than the colloidal gold test strip using the same CL-McAb. The preparedrare earth fluorescent test strip has good specificity, repeatability, accuracy with theshelf life at least six months.
Fluorescein isothiocyanate (FITC) with excellent luminescent properties had beenselected to prepare the FITC-SiO2-doped nanoparticles based on the silica nanoparticles by the St ber method. The FITC-SiO2-doped nanoparticles have the fluorescencecharacteristic peaks of FITC, good lightfastness and not prone to fluorescent leak. Theyhad been appeared good dispersion and uniform particle size of96±6nm withhomogeneous globular structure under TEM. The FITC-SiO2-doped nanoparticles withamino group had successfully couplied with CL-McAb through the aldehyde groups ofthe oxidized Dextran-500k. The FITC fluorescent antibody has benn sprayed on theconjugate pad. CL-BSA and SPA were selected as T line and C line blot of the NCmembrane. The sample pad, conjugate pad, NC membrane and the absorbent paper werefixed on the support plate according to a certain process to produce the FITCfluorescence test strip. The performance of the FITC fluorescent test strip had beenidentified. The limit of completely suppressed of the strip with naked-eye is6ng/mL,meanwhile quantitative detection range of test strip using the micro-fluorescenceanalyzer is0.16～5.18ng/mL with the LOD0.16ng/mL that is better than the colloidalgold test strip using the same CL-McAb. The prepared FITC fluorescent test strip hasgood specificity, repeatability, accuracy with the shelf life at least six months.
A GC-MS method to detect CL residue in pig urine sample had been established.The linear detection range of the GC-MS method is0.10~500ng/mL with the LOD0.1ng/mL. The method is sensitive, accurate, stability but requires expensive equipmentequipment, specialized operators, long testing cycle, cumbersome operation, high costand can not do on-site high-throughput testing. Rare earth fluorescent test strips andFITC fluorescence test strips were compared with GC-MS method by analysis the actualpig urine sample. The coincidence rate of the rare earth fluorescent test strips was100%compared with GC-MS method. The coincidence rate of the FITC fluorescent test stripswas97.6%compared with GC-MS method. All the results further confirmed that therare earth fluorescent test strips and FITC fluorescence test strips have good accuracyand timeliness in the detection of CL resude.
采用反相微乳法制备了SiO2纳米颗粒，通过改变DDW的量得到了粒径在14～97nm之间的分散性较好、粒径均一的球形SiO2纳米颗粒。同时采用St ber法制备了SiO2纳米颗粒，通过改变TEOS的量得到了粒径在93～398nm之间的单分散、粒径均一的球形SiO2纳米颗粒。用硅烷偶联剂APTS对制备的SiO2纳米颗粒进行有水条件下的表面修饰，用傅里叶红外光谱仪检测改性结果，证明改性成功。
选择具有优良发光性能的稀土配合物BHHCT-Eu3+为荧光材料，在反相微乳法合成SiO2的基础上，制备了共价型稀土荧光纳米颗粒，通过多次包壳，增加了荧光纳米颗粒的荧光强度。在TEM下，稀土荧光纳米颗粒呈现均匀的球状结构，粒径为59±5nm，具有较好的分散性。利用氧化后Dextran-500k的醛基成功的将带氨基的稀土荧光纳米颗粒和CL-McAb联接，将稀土荧光抗体喷涂在结合垫上，分别选择CL-BSA和SPA作为NC膜上的T线和C线印迹，将样品垫、结合垫、NC膜、吸水纸按照一定的工艺固定在支持板上，从而制成了稀土荧光检测试纸。对稀土荧光检测试纸的性能进行了鉴定，其肉眼观测完全抑制限为1.6ng/mL，通过微型荧光读条仪可以进行定量检测，定量检测范围为0.04～1.27ng/mL，LOD为0.04ng/mL，较同抗体制备的胶体金试纸灵敏许多，具有较好的特异性，与同系物几乎无交叉，具有良好的重复性、准确性，有效期至少为6个月。
选择具有优良发光性能的FITC为荧光材料，利用St ber法制备了掺杂型的FITC-SiO2纳米颗粒，该荧光纳米颗粒具有FITC的荧光特征峰，有较好的耐光性，不易发生荧光泄露。在TEM下，FITC-SiO2纳米颗粒呈现均匀的球状结构，粒径为96±6nm，具有较好的分散性。利用氧化后Dextran-500k的醛基将带氨基的FITC-SiO2纳米颗粒和CL-McAb联接，将FITC荧光抗体喷涂在结合垫上，分别选择CL-BSA和SPA作为NC膜上的T线和C线印迹，将样品垫、结合垫、NC膜、吸水纸按照一定的工艺固定在支持板上，从而制成了FITC荧光检测试纸。对FITC荧光检测试纸的性能进行了鉴定，其肉眼观测完全抑制限为6ng/mL，通过微型荧光读条仪可进行定量检测，定量检测范围为0.16～5.18ng/mL，LOD为0.16ng/mL，较同抗体制备的胶体金试纸灵敏，具有较好的特异性，与同系物几乎无交叉，具有良好的重复性、准确性，有效期至少为6个月。
建立了检测猪尿样品中CL残留的GC-MS方法，其线性检测范围为0.10～500ng/mL，LOD为0.1ng/mL，该法灵敏、准确、稳定，需要昂贵的仪器设备，专门的操作人员，检测周期长，费用较高，无法做到现场高通量检测。比较分析了GC-MS、稀土荧光试纸和FITC荧光试纸对实际样品的测定结果，稀土荧光检测试纸与确证方法GC-MS的符合率为100%，FITC荧光检测试纸与确证方法GC-MS的符合率为97.6%，进一步确证了稀土荧光试纸和FITC荧光试纸检测CL残留具有较好的准确性、时效性。
Two complete antigens of CL-BSA and CL-OVA were successfully prepared.BALB/c mice were immunized with CL-BSA, monoclonal antibody against CL(CL-McAb) was prepared by hybridoma technology. Three hybridoma cell lines werescreened. The hybridoma cell line1F8which the titer was1:1280in supernatant wereinoculated with liquid paraffin treated mice. CL-McAb ascites were collected and tested.The titer of hybridoma cell line1F8was1:512000of in ascites and the indirectcompetitive ELISAresults showed the half inhibitory concentration (IC50) of2.45ng/mLto CL. The affinity constant (Ka) of the CL-McAb was3.75×1010L/mol and thecross-reactivity with other drugs were less than0.08%. CL-McAb with high-titer, highsensitivity high affinity and high specificity had been produced successfully in thisexperiment.
Spherical silica nanoparticles with good dispersion and uniform particle sizebetween14and97nm were successfully abtained by varying the amount of water in thepreparation through inverse microemulsion method. Spherical silica nanoparticles withgood dispersion and uniform particle size between93and398nm were successfullyabtained by varying the amount of TEOS in the preparation through St ber method. Asilane coupling agent APTS was used to modify the surface of the silica nanoparticlesunder the conditions of in water. The Fourier transform infrared spectroscopy resultsprove that the silica nanoparticles have been successful modification.
Rare earth chelates BHHCT-Eu3+with excellent luminescent properties had beenselected to prepare the covalent type rare earth fluorescent nanoparticles based on thesilica nanoparticles by the reverse microemulsion method. The fluorescence intensity ofthe rare earth fluorescent nanoparticles increased through multiple cladding, and thefluorescent nanoparticles appeard good dispersion and uniform particle size of59±5nmwith homogeneous globular structures under TEM. The rare earth fluorescentnanoparticles with amino group had successfully couplied with CL-McAb through thealdehyde groups of the oxidized Dextran-500k. The rare earth fluorescent antibody hasbenn sprayed on the conjugate pad. CL-BSAand SPAwere selected as T line and C lineblot of the NC membrane. The sample pad, conjugate pad, NC membrane and theabsorbent paper were fixed on the support plate according to a certain process toproduce the rare earth fluorescence test strip. The performance of the rare earthfluorescent test strip had been identified. The limit of completely suppressed of the stripwith naked-eye is1.6ng/mL, meanwhile quantitative detection range of test strip usingthe micro-fluorescence analyzer is0.04~1.27ng/mL with the LOD0.04ng/mL that ismuch better than the colloidal gold test strip using the same CL-McAb. The preparedrare earth fluorescent test strip has good specificity, repeatability, accuracy with theshelf life at least six months.
Fluorescein isothiocyanate (FITC) with excellent luminescent properties had beenselected to prepare the FITC-SiO2-doped nanoparticles based on the silica nanoparticles by the St ber method. The FITC-SiO2-doped nanoparticles have the fluorescencecharacteristic peaks of FITC, good lightfastness and not prone to fluorescent leak. Theyhad been appeared good dispersion and uniform particle size of96±6nm withhomogeneous globular structure under TEM. The FITC-SiO2-doped nanoparticles withamino group had successfully couplied with CL-McAb through the aldehyde groups ofthe oxidized Dextran-500k. The FITC fluorescent antibody has benn sprayed on theconjugate pad. CL-BSA and SPA were selected as T line and C line blot of the NCmembrane. The sample pad, conjugate pad, NC membrane and the absorbent paper werefixed on the support plate according to a certain process to produce the FITCfluorescence test strip. The performance of the FITC fluorescent test strip had beenidentified. The limit of completely suppressed of the strip with naked-eye is6ng/mL,meanwhile quantitative detection range of test strip using the micro-fluorescenceanalyzer is0.16～5.18ng/mL with the LOD0.16ng/mL that is better than the colloidalgold test strip using the same CL-McAb. The prepared FITC fluorescent test strip hasgood specificity, repeatability, accuracy with the shelf life at least six months.
A GC-MS method to detect CL residue in pig urine sample had been established.The linear detection range of the GC-MS method is0.10~500ng/mL with the LOD0.1ng/mL. The method is sensitive, accurate, stability but requires expensive equipmentequipment, specialized operators, long testing cycle, cumbersome operation, high costand can not do on-site high-throughput testing. Rare earth fluorescent test strips andFITC fluorescence test strips were compared with GC-MS method by analysis the actualpig urine sample. The coincidence rate of the rare earth fluorescent test strips was100%compared with GC-MS method. The coincidence rate of the FITC fluorescent test stripswas97.6%compared with GC-MS method. All the results further confirmed that therare earth fluorescent test strips and FITC fluorescence test strips have good accuracyand timeliness in the detection of CL resude.
引文
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