氯嘧磺隆单克隆抗体的制备及其单链抗体基因的克隆

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1、中国农业大学博士学位论文氯嘧磺隆单克隆抗体的制备及其单链抗体基因的克隆姓名：羿国香申请学位级别：博士专业：作物栽培与耕作学指导教师：何钟佩;王保民20060601AbstractThisdissertationreportedtheproductionandspecificityofmonoclonalantibodyagainstchlorimuron．ethyl，theestablishmentofELISAandcolloidalgolddipstickforthedetectofchlorimuron．ethyl

2、basedonmonoclonalantibody,andthecloningofScFvgeneangainstchlorimuron·ethyl．Haptenwassynthesizedandverifiedbymassspectrometry(MS)．Then，thehaptenswerecovalenlyconjugatedtobovineserumalbumin(BSA)andovalbumin(OVA)toprepareantigenandcoatingantigen，respectively．Theconj

3、ugateswereidentifiedandtheirmolecularratioofhaptentocagierproteinweredeterminedbyUVspectrophotmetry．Balb／cfemalemicewereimmunizedwithsyntheticantigens．andtitersof04-1．6x104foranti—happen—BSAwereobtainedfromfiveofthem．Amonoclonalantibody(McAb)specificforchlorimuro

4、n-ethylwasproducedbyhybridomatechnology．ThespecificityofMcAbwascharacterizedbyELISA．TheresultsshowedthattheMcAbhadatiterof6．4x104andwasbelongtoIgGlsubclassandKisotype．Theaffinityconstantwas3．27x10’L／m01．TheMcAbwasofhighspecifictochlorimuron—ethylandhadlessthan2．2

5、％crossreactivitytochlorimuron-ethylanalogs．AnindirectcompetitiveELISA(iC—ELISA)andasimplifiedindirectcompetitiveELISA(simplifiediC-ELISA)weredevelopedbasedontheMcAb．TheiC—ELISAhadaIC50valueof11．6ng／mLandalinearworkingrangeofI．6-84ng／mLforstandardchlorimuron—ethyl

6、sample．Recoveryrangewasfrom74％to114％intapewater(1-500neflmL)andfrom216％to326％insoilf002—1．25I_tg／g)．ComparedwithiC-ELISA,thesimplifiediC—ELISAhadaIC50valueof28．7ng／mLandalinearworkingrangeof2．2-372ng／mLforstandardchlorimuron·ethylsample．Recoveryrangewasfrom84％-10

7、2％intapewaterandfrom99％to129％insoil．Obviously,thesimpleiC—ELISAhasbeaeraccuracythantheconventioniC—ELISA．Amonoclonalantibody-basedlateralflowdipstickwasdevelopedinwhichthedetectorreagentwascolloidalgold—labeledaffinitypurlfedmonoclonalanti—chlorimuron-ethylantibo

8、dy．Thecapturereagentishapten·bovineserumalbuminconjugateandgoatanti—mouseantibodywhichisimmobilizedontheNCmembrane．Thevisualdetectionlimitforchlorimuron-ethylw