Santella R M, Weston A, Perera F P, Trivers G T, Harris C C, Young T L, Nguyen D, Lee B M, Poirier M C
Cancer Center, School of Public Health, Columbia University, New York, NY 10032.
Carcinogenesis. 1988 Jul;9(7):1265-9. doi: 10.1093/carcin/9.7.1265.
An interlaboratory comparison of immunoassays using antisera elicited against benzo[a]pyrene-diol-epoxide-modified DNA (BPDE-I-DNA) was carried out resulting in standardization of antisera, competitors and assay conditions. The assays used included competitive enzyme-linked immunosorbent assays (ELISA) with color and fluorescence endpoint detection and an ultrasensitive enzyme radioimmunoassay (USERIA) with a radioactive endpoint. Three different antisera were compared, two of which were obtained from different rabbits immunized with the same BPDE-I-DNA and a third from an animal immunized with another BPDE-I-DNA sample. Samples of standardized BPDE-I-DNA with high (36 pmol adduct/microgram DNA; 1.2 adducts/10(2) nucleotides) and low (4.5 fmol/microgram DNA; 1.5 adducts/10(6) nucleotides) modification levels were prepared and used in each laboratory. The antisera were all elicited against DNAs modified to a high extent, and it was therefore not surprising that they detected adducts in a slightly modified DNA sample with lower efficiency than those in highly modified DNA samples. The discrepancy of antibody recognition between the highly and slightly modified samples varied between 1.4- and 11.2-fold depending on the antiserum and assay. To ascertain the quantitative capability of the immunoassays, the modification level of DNA isolated from mouse keratinocytes treated with [3H]benzo[a]pyrene was determined by radioactivity and immunoassay. These results indicated that when a biological sample is assayed against a BPDE-I-DNA standard modified in the same range as the biological samples (4.5 fmol/microgram), quantitative recovery of adducts is achieved by immunoassay. These studies resulted in the realization that interlaboratory differences in immunoassay procedure can have significant consequences for data comparison and that where possible it is preferable for laboratories to use the same antisera and modified DNA standards.
开展了一项实验室间比较，使用针对苯并[a]芘二醇环氧化物修饰DNA（BPDE-I-DNA）产生的抗血清进行免疫测定，从而实现了抗血清、竞争剂和测定条件的标准化。所使用的测定方法包括具有颜色和荧光终点检测的竞争性酶联免疫吸附测定（ELISA）以及具有放射性终点的超灵敏酶放射免疫测定（USERIA）。比较了三种不同的抗血清，其中两种是从用相同BPDE-I-DNA免疫的不同兔子获得的，第三种是从用另一种BPDE-I-DNA样品免疫的动物获得的。制备了具有高（36 pmol加合物/微克DNA；1.2个加合物/10(2)个核苷酸）和低（4.5 fmol/微克DNA；1.5个加合物/10(6)个核苷酸）修饰水平的标准化BPDE-I-DNA样品，并在每个实验室中使用。所有抗血清均针对高度修饰的DNA产生，因此毫不奇怪，它们检测轻度修饰DNA样品中的加合物的效率低于高度修饰DNA样品中的加合物。高度修饰和轻度修饰样品之间抗体识别的差异在1.4至11.2倍之间，具体取决于抗血清和测定方法。为了确定免疫测定的定量能力，通过放射性和免疫测定法测定了用[3H]苯并[a]芘处理的小鼠角质形成细胞中分离的DNA的修饰水平。这些结果表明，当针对与生物样品修饰范围相同（4.5 fmol/微克）的BPDE-I-DNA标准物测定生物样品时，免疫测定可实现加合物的定量回收。这些研究结果表明，免疫测定程序中的实验室间差异可能对数据比较产生重大影响，并且在可能的情况下，实验室最好使用相同的抗血清和修饰DNA标准物。
