斑蝥素提取、特异性抗体的制备及其酶联免疫检测方法的分析-extraction of cantharidin, preparation of specific antibody and analysis of enzyme-linked immunosorbent assay method.docxVIP

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斑蝥素提取、特异性抗体的制备及其酶联免疫检测方法的分析-extraction of cantharidin, preparation of specific antibody and analysis of enzyme-linked immunosorbent assay method.docx

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斑蝥素提取、特异性抗体的制备及其酶联免疫检测方法的分析-extraction of cantharidin, preparation of specific antibody and analysis of enzyme-linked immunosorbent assay method

ABSTRACTCantharidinistheactiveingredientofthecanthariswhichisTranditionalChineseMedicines.CantharidinisalsoonekindofnaturaldefensivetoxinsofTerpenoid.Therearealotofresearchesachievedabouttheanti-cancermechanisms,itspharmacologyorthetoxicology.Andalargenumberofcantharidinderivativesweresynthesized.However,tharearestillmanyproblemsunknown,suchasbiosynthesissitesorrutesandtheanticancermechanism.Atpresent,thecantharidin-detectionmethodssuchasweight,pH,UVspectrophotometry,HPLC,GC,areusuallynottooconvenient.Someofthemrequireexpensivechromatographyinstruments,someofthemrequirethesamplebepurifiedbeforebeingdetected,andsomeofthemevenrequesttheoperaterequiptthehightechlonogy.Objectives:Extractthecantharidinfromthecantharis,andbesedonthis,synthesiscantharidincarboxylderivativesN-carboxyethylcantharidimide.Andinquirethebestconditionsandthehighestyieldsforthereaction.Thencomparethetoxicityofthenewderivativestothatofcanthsridin.PreparetheantigenbycoupleingthehaptenN-carboxyethylcantharidimidetothecarrierprotein.Preparethespecificantibodybyimunningrabbits.Ultimately,undertheantigen-antibodyreactionprinciple,establishcantharidinindirectcompetitiveELISAmethodanditscurve.Methods:Extractcantharidinbymixedextractionmethod,shockingmethodandrapidextractionmethodrespectively.Aseriesreactionsofcantharidinwithβ-alaninewerecarriedoutwithsinglefactordiffered.DetectandidentifytheextractedandsynthesizedchemicalsbyGC,TLC,UV,MSandNMR.Andcomparethestomachpoisoningtoxicitybydeterminingthetwochemicals’LD50inmicewithmodifiedKabermethod.UsecoupleagentNHS,DCCtocoupletheN-carboxyethylcantharidimidetothecarrierproteinbyNHSestermethord,andtestorconfiemtheproductbymolarabsorptioncoefficientmethod.Then,immunerabbitstogetantibodyanddetecttheantibodytiterbyELISA.Whentheantibodytiterisabove1:3200,separatetheantiserum,then,purifytheantibodyintheantiserumwithCNBr-activatedagarosegel4Baffinitychromatography.Then,detectthepurifiedantibodybySDSandELISA.Usethepurifiedantibodyandtheantigentoestablishcantharidinindire