Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA)

B. Schoket, W. Alan Doty, István Vincze, Paul T. Strickland, Giovanni M. Ferri, Giorgio Assennato, Miriam C. Poirier

Research output: Contribution to journalArticle

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Abstract

A competitive enzyme-linked immunosorbent assay (ELISA), the most frequently used immunoassay for the determination of polycyclic aromatic hydrocarbonDNA adducts in human tissues, has been modified to achieve approximately a 6-fold increase in sensitivity. The new assay, a competitive dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) has utilized the same rabbit antiserum as the ELISA, antiserum elicited against DNA modified with benzo[a]pyrene. However, the alkaline phosphatase conjugate has been replaced with a biotin-europium-labeled streptavidin signal amplification system, and the release of europium into the solution forms a highly fluorescent chelate complex that is measured by time-resolved fluorometry. The DELFIA has achieved a 5- to 6-fold increase in sensitivity for measurement of DNA samples modified in vitro with benzo[a]pyrene, for cultured cells exposed to radiolabeled benzo[a]pyrene, and for human samples from occupationally exposed workers. The assay has been validated by comparison of adduct levels determined by DELFIA, ELISA, and radioactivity in DNA from mouse keratinocytes exposed to radiolabeled benzo[a]pyrene. Human lymphocyte DNA samples from 104 Hungarian aluminum plant workers were assayed by ELISA and compared to blood cell DNA samples from 69 Italian coke oven workers assayed by DELFIA. The standard curves demonstrated that the limit of detection of 4.0 adducts in 108 nucleotides for polycyclic aromatic hydrocarbon-DNA adducts by ELISA, using 35 Mg of DNA/microtiter plate well, has been decreased to 1.3 adducts in 108 nucleotides by DELFIA, using 20 μg of DNA/microtiter well. If 35 μg of DNA were used in the DELFIA, the calculated detection limit would be 0.7 adducts in 108 nucleotides. For the occupationally exposed groups presented here, the biologically effective dose, as evidenced by blood cell polycyclic aromatic hydrocarbon-DNA adduct levels, was clearly higher in the Hungarian aluminum plant workers; without the added sensitivity of the DELFIA many of the positive samples among the Italian coke oven workers would have been undetectable.

Original languageEnglish
Pages (from-to)349-353
Number of pages5
JournalCancer Epidemiology Biomarkers and Prevention
Volume2
Issue number4
Publication statusPublished - 1993

Fingerprint

Fluoroimmunoassay
Lanthanoid Series Elements
Benzo(a)pyrene
DNA
Enzyme-Linked Immunosorbent Assay
Coke
Europium
Nucleotides
Aluminum
Limit of Detection
Immune Sera
Blood Cells
Fluorometry
Streptavidin
polycyclic aromatic hydrocarbons-DNA adduct
Biotin
Keratinocytes
Immunoassay
Radioactivity
Alkaline Phosphatase

ASJC Scopus subject areas

  • Epidemiology
  • Oncology

Cite this

Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). / Schoket, B.; Alan Doty, W.; Vincze, István; Strickland, Paul T.; Ferri, Giovanni M.; Assennato, Giorgio; Poirier, Miriam C.

In: Cancer Epidemiology Biomarkers and Prevention, Vol. 2, No. 4, 1993, p. 349-353.

Research output: Contribution to journalArticle

Schoket, B. ; Alan Doty, W. ; Vincze, István ; Strickland, Paul T. ; Ferri, Giovanni M. ; Assennato, Giorgio ; Poirier, Miriam C. / Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). In: Cancer Epidemiology Biomarkers and Prevention. 1993 ; Vol. 2, No. 4. pp. 349-353.
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