BAM Federal Institute for Materials Research and Testing, Division 1.5, Protein Analysis, Richard-Willstätter-Strasse 11, 12489 Berlin, Germany.
J Mol Recognit. 2012 Feb;25(2):89-97. doi: 10.1002/jmr.2162.
Antibodies are a promising tool for the fast and selective trace detection of explosives. Unfortunately, the production of high-quality antibodies is not trivial and often expensive. Therefore, excellent antibodies are a rare and limiting resource in fields such as biosensing, environmental analysis, diagnostics, cancer therapy, and proteomics. Here, we report the synthesis, bioconjugation, and application of the structurally optimized hapten 6-(2,4,6-trinitro)-phenylhexanoic acid to improve the selectivity and sensitivity of antibodies for the detection of one of the most important explosives, trinitrotoluene. With a conjugate of bovine serum albumin and a highly purified N-hydroxy-succinimide (NHS)-activated hapten, two rabbits were immunized to obtain polyclonal antibodies. The immunization process was monitored by enzyme-linked immunosorbent assay to gain information about the progress of antibody titer and affinity. Finally, the polyclonal antibodies reached an affinity constant of (5.1 ± 0.6) × 10(9) l/mol (rabbit R1) and (2.3 ± 0.2) × 10(9) l/mol (rabbit R2). The respective assays show a minimum test midpoint (IC(50) value) of 0.1 ± 0.01 µg/l (R1) and 0.2 ± 0.02 µg/l (R2) and a working range of 0.005 to 150 µg/l (R1) and 0.007 to 200 µg/l (R2), which corresponds to more than four orders of magnitude for both. This is quite remarkable for a competitive immunoassay, which is often believed to have a narrow dynamic range. The limit of detection was calculated to 0.6 ng/l (R1) and 1.5 ng/l (R2), which is up to 100 times improvement in relation to the assay of Zeck et al. (1999) on the basis of a monoclonal antibody. The excellent selectivity of the polyclonal antibodies was comprehensively examined by determining the cross-reactivity to common explosives and other nitroaromatics including nitro musk components. The widely held belief that polyclonal antibodies generally display higher cross-reactivities than monoclonals could be disproved.
抗体是一种用于快速、选择性痕量检测爆炸物的有前途的工具。不幸的是，高质量抗体的生产并不简单，而且通常很昂贵。因此，在生物传感、环境分析、诊断、癌症治疗和蛋白质组学等领域，优秀的抗体是一种稀有且有限的资源。在这里，我们报告了结构优化的半抗原 6-(2,4,6-三硝基）-苯己酸的合成、生物偶联及其应用，以提高抗体对检测最重要的爆炸物之一三硝基甲苯的选择性和灵敏度。用牛血清白蛋白缀合物和高度纯化的 N-羟基琥珀酰亚胺（NHS）激活的半抗原免疫两只兔子，以获得多克隆抗体。通过酶联免疫吸附试验监测免疫过程，以获得有关抗体效价和亲和力进展的信息。最后，多克隆抗体的亲和常数达到（5.1±0.6）×10(9) l/mol（兔子 R1）和（2.3±0.2）×10(9) l/mol（兔子 R2）。各自的测定法显示出最小测试中点（IC(50) 值）为 0.1±0.01μg/l（R1）和 0.2±0.02μg/l（R2）以及工作范围为 0.005 至 150μg/l（R1）和 0.007 至 200μg/l（R2），这对于竞争免疫测定法来说是相当显著的，竞争免疫测定法通常被认为动态范围较窄。检测限计算为 0.6ng/l（R1）和 1.5ng/l（R2），与 Zeck 等人（1999 年）基于单克隆抗体的测定法相比提高了 100 倍。通过测定常见爆炸物和其他包括硝基麝香成分在内的硝基芳烃的交叉反应性，全面检查了多克隆抗体的优异选择性。普遍认为多克隆抗体通常比单克隆抗体显示出更高的交叉反应性，但这种观点可能是错误的。
