Scholler Nathalie, Lowe Kimberly A, Bergan Lindsay A, Kampani Archana V, Ng Vivian, Forrest Robin M, Thorpe Jason D, Gross Jenny A, Garvik Barbara M, Drapkin Ronny, Anderson Garnet L, Urban Nicole
School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 96104-6069, USA.
Clin Cancer Res. 2008 May 1;14(9):2647-55. doi: 10.1158/1078-0432.CCR-07-1442.
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.
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.
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).
Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead-based assays for use in large validation studies.
为了检测循环抗原，夹心酶联免疫吸附测定（ELISA）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，但由于其生产过程漫长且成本高昂，重组抗体正成为一种有吸引力的替代选择。
我们开发了一类名为生物抗体（Bb）的新型重组抗体，并将其与用于血清诊断的单克隆抗体进行比较。Bb由二倍体酵母在体内分泌生物素化，并在镍纯化后用作亲和试剂。使用Bb与多克隆抗体组合开发了基于磁珠的HE4和间皮素检测方法（Bb/pAb检测方法）。为了评估精密度，在每个标志物的六个分析物水平上进行了四次运行，每次运行16个重复的重复性研究。在214份患者血清样本中进行了Pearson相关性分析和受试者工作特征分析，以直接将Bb/pAb检测方法与单克隆抗体检测方法进行比较。在一组扩大的336例卵巢癌病例和对照中进一步评估了Bb/pAb检测方法的诊断性能。
在分析物水平上平均而言，Bb/pAb检测方法对HE4的批内和批间变异系数分别为11.7和23.8，对间皮素分别为14.0和14.5。在子集（n = 214）中，单克隆抗体和Bb/pAb检测方法之间对HE4的Pearson相关性为0.95，对间皮素为0.92。单克隆抗体和Bb/pAb检测方法对HE4的曲线下面积无显著差异（分别为0.88和0.84；P = 0.20），对间皮素也无显著差异（分别为0.74和0.72；P = 0.38）。
酵母分泌的Bb可可靠地用于经济高效且高度灵敏的基于磁珠的检测方法，用于大型验证研究。
