A Novel Fluorescence Immunoassay System Based on pH-Sensitive Phase Separating Technique-中国期刊网

摘要 Inthispaper,itwasdiscoveredthatanovelpH-sensitivecopolymerofN-isopropylacrylamide(NIP)andN-(3-dimethylaminopropyl)methacrylamide(DMAPM)couldbegottenbypolymerization.ThephasetransitionpH(pHtr)ofP(NIP-DMAPM)polymerwasfoundtobe7.4at37℃.ThepolymerwasprecipitatedoutofwateraboveacriticalpH=7.4andre-dissolvedbelowpH----7.4.Thecharacteristicofthispolymermadeitpossibletocarryouttheimmunochemicalstepsofanimmunoassayinatruesolutionandthentoquicklyseparatetheresultingproductfromthereactionmixture.Inacompetitivefluorescenceimmunoassay,thestandardrabbitIgGandrabbitIgGimmobilizedonP(NIP-DMAPM)firstcompetitivelyreactedwiththefluoresceinisothiocyanate(FITC)labeledantibody,thenthepHofsolutionwasadjustedabovethepHtrofpolymertoprecipitatethepolymer-immunecomplex,andthepolymer-immunecomplexprecipitatewasseparatedandre-dissolvedbytheadjustmentofpH,finallytheFITC-labeledantibodyintheimmunecomplexwasquantifiedbyfluorescencemeasurement.ThecalibrationgraphforrabbitIgGwaslinearovertherangeof100-1000ng/mLwithadetectionlimitof11ng/mL.Themethodisrapid,sensitiveandsimple.OwingtoneutralpHtrofP(NIP-DMAPM),thedamagetoantigen-antibodyimmunecomplexwasgreatlydecreasedinthecourseofseparation.Inaddition,asandwichenzyme-linkedfluorescenceimmunoassaymethodforthedeterminationofhumanIgGwasalsodeveloped,showingthatthepH-sensitivephaseseparatingimmunoassaycouldbeperformedinthecompetitivemethodaswellasthesandwichmethod.

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A Novel Fluorescence Immunoassay System Based on pH-Sensitive Phase Separating Technique

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A Novel Fluorescence Immunoassay System Based on pH-Sensitive Phase Separating Technique

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