Seurynck-Servoss Shannon L, Baird Cheryl L, Miller Keith D, Pefaur Noah B, Gonzalez Rachel M, Apiyo David O, Engelmann Heather E, Srivastava Sudhir, Kagan Jacob, Rodland Karin D, Zangar Richard C
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99354, USA.
Proteomics. 2008 Jun;8(11):2199-210. doi: 10.1002/pmic.200701036.
Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust-affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single-chain antibodies (scFv) are affinity reagents that have greater potential for high-throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope-tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate-specific antigen (PSA) are highly specific when tested against 21 different IgG-based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R(2) = 0.95) to a commercial 96-well ELISA when tested using human serum samples. In addition, we find that TRX-scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.
夹心酶联免疫吸附测定（ELISA）微阵列在验证疾病生物标志物方面具有巨大潜力。每个ELISA都依赖于强大的亲和试剂，这些试剂在固定于固体表面或标记用于检测时仍保持活性。单链抗体（scFv）是一种亲和试剂，与传统的IgG相比，具有更大的高通量生产潜力。不幸的是，scFv在固定于固体表面后通常比IgG活性更低，并不总是适用于夹心ELISA。因此，我们研究了不同的固定策略和scFv构建体，以确定一种更强大的将scFv用作ELISA试剂的策略。这些研究中出现了两种有前景的策略：（i）使用抗表位抗体预捕获表位标记的scFv；（ii）在载玻片上直接打印硫氧还蛋白（TRX）/scFv融合蛋白。尽管抗表位预捕获方法存在试剂转移的风险，但这两种策略都提高了固定化scFv的稳定性，并提高了scFv ELISA微阵列测定的灵敏度。使用直接打印方法，我们发现针对前列腺特异性抗原（PSA）的scFv在针对21种不同的基于IgG的测定进行测试时具有高度特异性。此外，当使用人血清样本进行测试时，scFv微阵列PSA测定给出了与商业96孔ELISA相当的定量结果（R² = 0.95）。此外，我们发现针对表皮生长因子和毒素X的TRX-scFv融合蛋白具有良好的检测限。总体而言，这些结果表明，对scFv构建体进行微小修饰足以产生适用于多重检测系统的试剂。
