Development of Methods for the Production of Monoclonal Antibodies Specific for Depurination Adducts of Benzo[a]pyrene and for Their Use in a Competitive Enzyme-linked Immunosorbent Assay

George P Casale, Rosa Todorovic, N. V S Ramakrishna, Eleanor G Rogan, Ercole Cavalieri

Research output: Contribution to journalArticle

Abstract

Nearly 80% of benzo[a]pyrene (BP)-DNA adducts are lost by depurination and excreted in the urine. These adducts offer a unique opportunity for biomonitoring BP exposure, although their hydrophobic nature poses challenges for methods development. We have implemented a competitive enzyme-linked immunosorbent assay (ELISA) for detection of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurination adduct of BP. Linkage of the adduct to keyhole limpet hemocyanin and bovine serum albumin via the heterobifunctional linker succinimidyl-4-(N-maleimido-methyl)-cyclohexane-l-carboxylate produced an effective immunogen and capture complex, respectively. Two cycles of hybridoma production yielded 23 monoclonal antibodies (MAb) specific for the capture complex. One of these MAb was used to optimize the competitive ELISA for BP-6-N7Gua. Conditions of the reaction between specific MAb and the free adduct were extensively modified to produce an assay that detects 50 fmol free adduct/mL reaction mixture.

Original languageEnglish (US)
Pages (from-to)53-61
Number of pages9
JournalPolycyclic Aromatic Compounds
Volume6
Issue number1-4
DOIs
StatePublished - 1994

Fingerprint

Immunosorbents
Monoclonal antibodies
Benzo(a)pyrene
Pyrene
Assays
Enzymes
Monoclonal Antibodies
DNA Adducts
Cyclohexane
Bovine Serum Albumin
7-(benzo(a)pyren-6-yl)guanine

Keywords

  • benzo[a]pyrene
  • DNA adducts
  • ELISA
  • MAb

ASJC Scopus subject areas

  • Polymers and Plastics
  • Materials Chemistry
  • Organic Chemistry

Cite this

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title = "Development of Methods for the Production of Monoclonal Antibodies Specific for Depurination Adducts of Benzo[a]pyrene and for Their Use in a Competitive Enzyme-linked Immunosorbent Assay",
abstract = "Nearly 80{\%} of benzo[a]pyrene (BP)-DNA adducts are lost by depurination and excreted in the urine. These adducts offer a unique opportunity for biomonitoring BP exposure, although their hydrophobic nature poses challenges for methods development. We have implemented a competitive enzyme-linked immunosorbent assay (ELISA) for detection of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurination adduct of BP. Linkage of the adduct to keyhole limpet hemocyanin and bovine serum albumin via the heterobifunctional linker succinimidyl-4-(N-maleimido-methyl)-cyclohexane-l-carboxylate produced an effective immunogen and capture complex, respectively. Two cycles of hybridoma production yielded 23 monoclonal antibodies (MAb) specific for the capture complex. One of these MAb was used to optimize the competitive ELISA for BP-6-N7Gua. Conditions of the reaction between specific MAb and the free adduct were extensively modified to produce an assay that detects 50 fmol free adduct/mL reaction mixture.",
keywords = "benzo[a]pyrene, DNA adducts, ELISA, MAb",
author = "Casale, {George P} and Rosa Todorovic and Ramakrishna, {N. V S} and Rogan, {Eleanor G} and Ercole Cavalieri",
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AU - Ramakrishna, N. V S

AU - Rogan, Eleanor G

AU - Cavalieri, Ercole

PY - 1994

Y1 - 1994

N2 - Nearly 80% of benzo[a]pyrene (BP)-DNA adducts are lost by depurination and excreted in the urine. These adducts offer a unique opportunity for biomonitoring BP exposure, although their hydrophobic nature poses challenges for methods development. We have implemented a competitive enzyme-linked immunosorbent assay (ELISA) for detection of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurination adduct of BP. Linkage of the adduct to keyhole limpet hemocyanin and bovine serum albumin via the heterobifunctional linker succinimidyl-4-(N-maleimido-methyl)-cyclohexane-l-carboxylate produced an effective immunogen and capture complex, respectively. Two cycles of hybridoma production yielded 23 monoclonal antibodies (MAb) specific for the capture complex. One of these MAb was used to optimize the competitive ELISA for BP-6-N7Gua. Conditions of the reaction between specific MAb and the free adduct were extensively modified to produce an assay that detects 50 fmol free adduct/mL reaction mixture.

AB - Nearly 80% of benzo[a]pyrene (BP)-DNA adducts are lost by depurination and excreted in the urine. These adducts offer a unique opportunity for biomonitoring BP exposure, although their hydrophobic nature poses challenges for methods development. We have implemented a competitive enzyme-linked immunosorbent assay (ELISA) for detection of 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurination adduct of BP. Linkage of the adduct to keyhole limpet hemocyanin and bovine serum albumin via the heterobifunctional linker succinimidyl-4-(N-maleimido-methyl)-cyclohexane-l-carboxylate produced an effective immunogen and capture complex, respectively. Two cycles of hybridoma production yielded 23 monoclonal antibodies (MAb) specific for the capture complex. One of these MAb was used to optimize the competitive ELISA for BP-6-N7Gua. Conditions of the reaction between specific MAb and the free adduct were extensively modified to produce an assay that detects 50 fmol free adduct/mL reaction mixture.

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