Identification of optimal conditions for the detection of benzo[a]pyrene-DNA adducts by enzyme-linked immunoadsorbent assays (ELISA)

F. Bucci, R. Galati, R. Zito, G. Falasca, A. Federico, A. Verdina

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The aim of the present report was to establish the optimal conditions for the detection of polycyclic aromatic hydrocarbon adducted to DNA by enzyme-linked immunoadsorbent assays (ELISA). Racemic 7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene ((±)-anti-BPDE) modified DNA samples were produced in vitro, by reacting (±)-anti-BPDE with calf thymus DNA, and in vivo in Swiss female mice by single i.p. injection of benzo[a]pyrene (B[a]P) (200 mg/kg body weight dissolved in tricaprylin). The BPDE adduct content in vitro and in liver and lung modified DNA was detected by direct and competitive ELISA using serial dilutions of the samples in unmodified calf thymus DNA, and polyclonal rabbit immunoglobulin-G elicited toward BPDE-DNA and BPDE-gelatin, both produced in our laboratory. The carcinogen-macromolecule conjugate in which adducts were sought could be used as an immunogen to produce a specific and potent antibody. Moreover, the modification level of the ELISA standards should be as close to the range as of the biological samples to correctly calculate the adducts, since different binding efficiency between antibody and BPDE-modified DNA is dependent on the BPDE modification level. Appropriate extraction of the in vitro modified samples is also necessary to guarantee the exact covalent modification level, eliminating noncovalently linked BPDE. Under these conditions, our results confirm that competitive ELISA is much more sensitive than the direct method, mainly because of the limitations caused by the coating of the antigen in each well (max 5 μg DNA/well), whereas the amount of DNA (modified or not) that can be employed for adduct detection by competitive ELISA increases 20-fold. The sensitivity obtained was 0.5 fmol B[a]P/μg DNA (1.6 adducts/107 nucleotides).

Original languageEnglish
Pages (from-to)2669-2674
Number of pages6
JournalAnticancer Research
Volume18
Issue number4 A
Publication statusPublished - Jul 1998

Fingerprint

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide
Immunosorbents
Benzo(a)pyrene
Enzymes
DNA
DNA Adducts
Antibodies
Polycyclic Aromatic Hydrocarbons
Gelatin
Carcinogens
Nucleotides
Immunoglobulin G
Body Weight
benzo(a)pyrene-DNA adduct
Rabbits
Antigens
Lung
Injections
Liver
In Vitro Techniques

Keywords

  • Benzo[a]pyrene
  • Carcinogenesis
  • DNA adducts
  • ELISA

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Identification of optimal conditions for the detection of benzo[a]pyrene-DNA adducts by enzyme-linked immunoadsorbent assays (ELISA). / Bucci, F.; Galati, R.; Zito, R.; Falasca, G.; Federico, A.; Verdina, A.

In: Anticancer Research, Vol. 18, No. 4 A, 07.1998, p. 2669-2674.

Research output: Contribution to journalArticle

Bucci, F. ; Galati, R. ; Zito, R. ; Falasca, G. ; Federico, A. ; Verdina, A. / Identification of optimal conditions for the detection of benzo[a]pyrene-DNA adducts by enzyme-linked immunoadsorbent assays (ELISA). In: Anticancer Research. 1998 ; Vol. 18, No. 4 A. pp. 2669-2674.
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