Use of yeast-secreted in vivo biotinylated recombinant antibodies (biobodies) in bead-based ELISA
Use of yeast-secreted in vivo biotinylated recombinant antibodies (biobodies) in bead-based ELISA
复制标题
DOI：
10.1158/1078-0432.ccr-07-1442
复制
发表时间：
2008-05-01
影响因子：
11.5
通讯作者：
Urban, Nicole
中科院分区：
文献类型：
--
作者：
Scholler, Nathalie;Lowe, Kirnberly A.;Urban, Nicole
目的：夹心ELISA法检测循环抗原需要两种互补的亲和试剂。小鼠单克隆抗体（mAb）和多克隆抗体（pAb）是常用的抗体，但由于其生产时间长且成本高，重组抗体正成为一种有吸引力的替代方案。实验设计：我们开发了一类新的重组抗体，称为生物体（Bb），并将其与mAb进行了血清诊断比较。利用二倍体酵母在体内进行生物素化，提纯后作为亲和试剂使用。利用Bbs与pAb （Bb/pAb测定）联合开发了基于珠状体的HE4和间皮素测定方法。为了评估准确性，对每个标记物在6个分析物水平上进行了4次16个重复的重复性研究。对214例患者血清样本进行Pearson相关性和受体-操作者特征分析，直接比较Bb/pAb和mAb检测结果。在336例卵巢癌病例和对照组中进一步评估了Bb/pAb检测的诊断性能。结果：在分析物水平上，Bb/pAb测定的运行内和运行间变异系数分别为HE4的11.7和23.8，间皮素的14.0和14.5。在子集（n = 214）中，mAb和Bb/pAb检测中HE4和间皮素的Pearson相关性分别为0.95和0.92。mAb和Bb/pAb测定HE4（分别为0.88和0.84,P = 0.20）和mesothelin（分别为0.74和0.72,P = 0.38）的曲线下面积无显著差异。结论：酵母分泌的Bbs可以可靠地用于具有成本效益且高灵敏度的基于头的分析，用于大型验证研究。
Purpose: To measure circulating antigens, sandwich ELISA assays require two complementary affinity reagents. Mouse monoclonal antibodies (mAb) and polyclonal antibodies (pAb) are commonly used, but because their production is lengthy and costly, recombinant antibodies are emerging as an attractive alternative.Experimental Design: We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays), To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls.Results: On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38).Conclusion: Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead - based assays for use in large validation studies.