Immobilization strategies for single-chain antibody microarrays
Immobilization strategies for single-chain antibody microarrays
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DOI：
10.1002/pmic.200701036
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发表时间：
2008-06-01
期刊：
影响因子：
3.4
通讯作者：
Zangar, Richard C.
中科院分区：
文献类型：
--
作者：
Seurynck-Servoss, Shannon L.;Baird, Cheryl L.;Zangar, Richard C.
夹心ELISA微阵列具有验证疾病生物标志物的巨大潜力。每种ELISA都依赖于固定在固体表面上或标记用于检测时保持活性的稳健亲和试剂。单链抗体（scFv）是亲和试剂，具有比传统IgG更大的高通量生产潜力。不幸的是，在固定在固体表面上之后，scFv的活性通常低于IgG，并且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，以确定使用scFv作为ELISA试剂的更稳健的策略。从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和（ii）在载玻片上直接印刷硫氧还蛋白（TRX）/scFv融合蛋白。这两种策略都提高了固定化scFv的稳定性，并增加了scFv ELISA微阵列测定的灵敏度，尽管抗表位预捕获方法引入了试剂转移的风险。使用直接打印方法，我们表明，抗前列腺特异性抗原（PSA）的scFv是高度特异性的，当测试对21个不同的IgG为基础的测定。此外，当使用人血清样品测试时，scFv微阵列PSA测定给出了与商业96孔ELISA相当的定量结果（R-2 = 0.95）。此外，我们发现针对表皮生长因子和毒素X的TRX-scFv融合物具有良好的LOD。总之，这些结果表明，scFv构建体的微小修饰足以产生适用于多重测定系统的试剂。
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.