Schoket B, Doty W A, Vincze I, Strickland P T, Ferri G M, Assennato G, Poirier M C
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH Bethesda, Maryland 20892.
Cancer Epidemiol Biomarkers Prev. 1993 Jul-Aug;2(4):349-53.
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)
一种竞争性酶联免疫吸附测定法（ELISA）是测定人体组织中多环芳烃 - DNA加合物最常用的免疫测定法，现已进行了改进，使其灵敏度提高了约6倍。新的测定法是一种竞争性解离增强镧系元素荧光免疫测定法（DELFIA），它使用了与ELISA相同的兔抗血清，该抗血清是针对用苯并[a]芘修饰的DNA产生的。然而，碱性磷酸酶偶联物已被生物素 - 铕标记的链霉亲和素信号放大系统所取代，铕释放到溶液中形成高度荧光螯合物，通过时间分辨荧光法进行测量。对于用苯并[a]芘体外修饰的DNA样品、暴露于放射性标记苯并[a]芘的培养细胞以及职业暴露工人的人体样品，DELFIA在测量DNA加合物时灵敏度提高了5至6倍。通过比较DELFIA、ELISA以及暴露于放射性标记苯并[a]芘的小鼠角质形成细胞DNA中的放射性所测定的加合物水平，对该测定法进行了验证。用ELISA对104名匈牙利铝厂工人的人淋巴细胞DNA样品进行了检测，并与用DELFIA检测的69名意大利炼焦炉工人的血细胞DNA样品进行了比较。标准曲线表明，使用35微克DNA/微孔板孔的ELISA检测多环芳烃 - DNA加合物时，10⁸个核苷酸中4.0个加合物的检测限，在用DELFIA、使用20微克DNA/微孔时已降至10⁸个核苷酸中1.3个加合物。如果在DELFIA中使用35微克DNA，计算出的检测限将为10⁸个核苷酸中0.7个加合物。（摘要截短于250字）
