title: Probing cocaine-antibody interactions in buffer and human serum.
abstract: BACKGROUND
  Despite progress in cocaine immunotherapy, the kinetic and thermodynamic properties of antibodies which bind to cocaine and its metabolites are not well understood. It is also not clear how the interactions between them differ in a complex matrix such as the serum present in the human body. In the present study, we have used microscale thermophoresis (MST), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) we have evaluated the affinity properties of a representative mouse monoclonal (mAb08) as well as those of polyclonal antibodies purified from vaccinated mouse and human patient serum.
  
  
  RESULTS
  MST analysis of fluorescently tagged mAb08 binding to cocaine reveals an approximately 15 fold decrease in its equilibrium dissociation constant in 20-50% human serum compared with that in saline buffer. A similar trend was also 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 both cocaine and the hapten used to raise these antibodies. The ITC data also revealed that cocaine has a moderate affinity of about 2 µM to 20% human serum and very little interaction with human serum albumin or nonspecific human IgG at that concentration range. In a SPR inhibition experiment, the binding of mAb08 to immobilized BSA-SNC was inhibited by cocaine and benzoylecgonine in a highly competitive manner, whereas the purified polyclonal antibodies from vaccinated humans and mice, revealed preferential selectivity to pharmacologically active cocaine but not to the inactive metabolite benzoylecgonine. We have also developed a simple binding model to simulate the challenges associated with cocaine immunotherapy using the variable quantitative and kinetic properties of the antibodies.
  
  
  CONCLUSIONS
  High sensitivity calorimetric determination of antibody binding to cocaine and its metabolites provide valuable information for characterization of their interactions and thermodynamic properties. In addition MST measurements of antibody affinity in the presence of biological fluids will provide a better opportunity to make reliable decisions and facilitate the design of cocaine vaccines and immunization conditions. The methods should be more widely adopted in characterization of antibody complexes.
authors.affiliation: Veterans Affairs Medical Center, Houston, Texas, United States of America.
authors.forename: Muthu
authors.initials: M
authors.lastname: Ramakrishnan
authors.forename: Fernando
authors.initials: F
authors.lastname: Alves De Melo
authors.forename: Berma M
authors.initials: BM
authors.lastname: Kinsey
authors.forename: John E
authors.initials: JE
authors.lastname: Ladbury
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Kosten
authors.forename: Frank M
authors.initials: FM
authors.lastname: Orson
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry* / Q000276:immunology; D000915:Antibody Affinity; D001667:Binding, Competitive; D002151:Calorimetry; D002417:Cattle; D003042:Cocaine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology*; D019970:Cocaine-Related Disorders / Q000276:immunology / Q000628:therapy; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry; D007167:Immunotherapy; D007700:Kinetics; D051379:Mice; D011485:Protein Binding; D044967:Serum / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D020349:Surface Plasmon Resonance
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D055767:Immobilized Proteins; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; C005618:benzoylecgonine; C066444:cocaethylene; D003042:Cocaine