Increased sensitivity for determination of polycyclic aromatic hydrocarbon-DNA adducts in human DNA samples by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA).
A competitive enzyme-linked immunosorbent assay (ELISA), the most frequently used immunoassay for the determination of polycyclic aromatic hydrocarbon-DNA 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 10(8) nucleotides for polycyclic aromatic hydrocarbon-DNA adducts by ELISA, using 35 micrograms of DNA/microtiter plate well, has been decreased to 1.3 adducts in 10(8) nucleotides by DELFIA, using 20 micrograms of DNA/microtiter well. If 35 micrograms of DNA were used in the DELFIA, the calculated detection limit would be 0.7 adducts in 10(8) nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)
Schoket
B
B
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH Bethesda, Maryland 20892.
Doty
W A
WA
Vincze
I
I
Strickland
P T
PT
Ferri
G M
GM
Assennato
G
G
Poirier
M C
MC
D000535:Aluminum; D001426:Bacterial Proteins; D001564:Benzo(a)pyrene; D002617:Chemical Industry; D003077:Coke; D004247:DNA / Q000032:analysis*; D003849:Deoxyguanosine / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005063:Europium; D005453:Fluorescence; D015200:Fluoroimmunoassay / Q000379:methods*; D005470:Fluorometry; D006801:Humans; D006814:Hungary; D007558:Italy; D008214:Lymphocytes / Q000378:metabolism; D008674:Metals, Rare Earth; D011083:Polycyclic Compounds / Q000032:analysis*; D019809:Streptavidin
D001426:Bacterial Proteins; D003077:Coke; D008674:Metals, Rare Earth; D011083:Polycyclic Compounds; D001564:Benzo(a)pyrene; D005063:Europium; D004247:DNA; D019809:Streptavidin; D000535:Aluminum; D003849:Deoxyguanosine