Probing Cocaine-Antibody Interactions in Buffer and Human Serum 英文参考文献.docVIP

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Probing Cocaine-Antibody Interactions in Buffer and Human Serum 英文参考文献.doc

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Probing Cocaine-Antibody Interactions in Buffer and Human Serum 英文参考文献

ProbingCocaine-AntibodyInteractionsinBufferand HumanSerum MuthuRamakrishnan1,2,4,FernandoAlvesDeMelo6,7,BermaM.Kinsey1,2,JohnE.Ladbury6,7 ThomasR.Kosten1,5,FrankM.Orson1,2,3,4 , * 1VeteransAffairsMedicalCenter,Houston,Texas,UnitedStatesofAmerica,2DepartmentofInternalMedicine,BaylorCollegeofMedicine,Houston,Texas,UnitedStates ofAmerica,3DepartmentofPathology,BaylorCollegeofMedicine,Houston,Texas,UnitedStatesofAmerica,4DepartmentofImmunologyAllergyandRheumatology, BaylorCollegeofMedicine,Houston,Texas,UnitedStatesofAmerica,5DepartmentofPsychiatry,BaylorCollegeofMedicine,Houston,Texas,UnitedStatesofAmerica, 6Department of Biochemistry and Molecular Biology, MD Anderson Cancer Center, University of Texas, Houston, Texas, United States of America, 7Center for BiomolecularStructureandFunction,MDAndersonCancerCenter,UniversityofTexas,Houston,Texas,UnitedStatesofAmerica Abstract Background:Despiteprogressincocaineimmunotherapy,thekineticandthermodynamicpropertiesofantibodieswhich bindtococaineanditsmetabolitesarenotwellunderstood.Itisalsonotclearhowtheinteractionsbetweenthemdifferin a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis(MST),isothermaltitrationcalorimetry(ITC),andsurfaceplasmonresonance(SPR)wehaveevaluatedthe affinitypropertiesofarepresentativemousemonoclonal(mAb08)aswellasthoseofpolyclonalantibodiespurifiedfrom vaccinatedmouseandhumanpatientserum. Results:MSTanalysisoffluorescentlytaggedmAb08bindingtococainerevealsanapproximately15folddecreaseinits equilibrium dissociation constantin 20–50%humanserum comparedwiththat insaline buffer. Asimilar trendwasalso found using enriched polyclonal antibodies purified from vaccinated mice and patient serum, for which we have used fluorescently tagged bovine serum albumin conjugated to succinyl norcocaine (BSA-SNC). This conjugate closely mimics bothcocaineandthehaptenusedtoraisetheseantibodies.TheITCdataalsorevealedthatcocainehasamoderateaffinity o