Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons
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Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons. / Georgiadis, Panagiotis; Kovács, Katalin; Kaila, Stella; Makedonopoulou, Paraskevi; Anna, Livia; Poirier, Miriam C; Knudsen, Lisbeth E; Schoket, Bernadette; Kyrtopoulos, Soterios A.
In: Mutagenesis, Vol. 27, No. 5, 2012, p. 589-97.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons
AU - Georgiadis, Panagiotis
AU - Kovács, Katalin
AU - Kaila, Stella
AU - Makedonopoulou, Paraskevi
AU - Anna, Livia
AU - Poirier, Miriam C
AU - Knudsen, Lisbeth E
AU - Schoket, Bernadette
AU - Kyrtopoulos, Soterios A
PY - 2012
Y1 - 2012
N2 - We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 µg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.
AB - We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 µg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.
U2 - 10.1093/mutage/ges024
DO - 10.1093/mutage/ges024
M3 - Journal article
C2 - 22610669
VL - 27
SP - 589
EP - 597
JO - Mutagenesis
JF - Mutagenesis
SN - 0267-8357
IS - 5
ER -
ID: 41851597