Cargando…
Probing Cocaine-Antibody Interactions in Buffer and Human Serum
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 bo...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409241/ https://www.ncbi.nlm.nih.gov/pubmed/22859949 http://dx.doi.org/10.1371/journal.pone.0040518 |
_version_ | 1782239567493988352 |
---|---|
author | Ramakrishnan, Muthu Alves De Melo, Fernando Kinsey, Berma M. Ladbury, John E. Kosten, Thomas R. Orson, Frank M. |
author_facet | Ramakrishnan, Muthu Alves De Melo, Fernando Kinsey, Berma M. Ladbury, John E. Kosten, Thomas R. Orson, Frank M. |
author_sort | Ramakrishnan, Muthu |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3409241 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34092412012-08-02 Probing Cocaine-Antibody Interactions in Buffer and Human Serum Ramakrishnan, Muthu Alves De Melo, Fernando Kinsey, Berma M. Ladbury, John E. Kosten, Thomas R. Orson, Frank M. PLoS One Research Article 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. Public Library of Science 2012-07-10 /pmc/articles/PMC3409241/ /pubmed/22859949 http://dx.doi.org/10.1371/journal.pone.0040518 Text en This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. https://creativecommons.org/publicdomain/zero/1.0/ This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration, which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. |
spellingShingle | Research Article Ramakrishnan, Muthu Alves De Melo, Fernando Kinsey, Berma M. Ladbury, John E. Kosten, Thomas R. Orson, Frank M. Probing Cocaine-Antibody Interactions in Buffer and Human Serum |
title | Probing Cocaine-Antibody Interactions in Buffer and Human Serum |
title_full | Probing Cocaine-Antibody Interactions in Buffer and Human Serum |
title_fullStr | Probing Cocaine-Antibody Interactions in Buffer and Human Serum |
title_full_unstemmed | Probing Cocaine-Antibody Interactions in Buffer and Human Serum |
title_short | Probing Cocaine-Antibody Interactions in Buffer and Human Serum |
title_sort | probing cocaine-antibody interactions in buffer and human serum |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3409241/ https://www.ncbi.nlm.nih.gov/pubmed/22859949 http://dx.doi.org/10.1371/journal.pone.0040518 |
work_keys_str_mv | AT ramakrishnanmuthu probingcocaineantibodyinteractionsinbufferandhumanserum AT alvesdemelofernando probingcocaineantibodyinteractionsinbufferandhumanserum AT kinseybermam probingcocaineantibodyinteractionsinbufferandhumanserum AT ladburyjohne probingcocaineantibodyinteractionsinbufferandhumanserum AT kostenthomasr probingcocaineantibodyinteractionsinbufferandhumanserum AT orsonfrankm probingcocaineantibodyinteractionsinbufferandhumanserum |