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Study on Fluorescent Immunochromatographic Stirp Based on Fluorescent Nano-particles Labeled for Detection of Clenbuterol Hydrochloirde

Author: SongChunMei
Tutor: ZhangGaiPing
School: Jiangnan University
Course: Food Nutrition and Safety
Keywords: Rare earths Fluorescein isothiocyanate Immunochromatographic teststrips Clenbuterol
CLC: TS207.53
Type: PhD thesis
Year: 2013
Downloads: 114
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Abstract

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.

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