The identification of a novel antibody for CD133 using human antibody phage display.
The identification of a novel antibody for CD133 using human antibody phage display.
复制标题
DOI：
10.1002/pros.23656
复制
发表时间：
2018-09
期刊：
影响因子：
--
通讯作者：
LeBeau AM
中科院分区：
文献类型：
--
作者：
Glumac PM;Forster CL;Zhou H;Murugan P;Gupta S;LeBeau AM
跨膜糖蛋白CD 133被认为是成人前列腺干细胞和癌症干细胞/起始细胞的标志物。研究CD 133在前列腺和癌症的正常生物学中的作用由于缺乏用于其检测的灵敏和准确的抗体而变得复杂。在这里，我们描述了一种独特的抗体的特性，确定使用人抗体噬菌体展示，可以识别CD 133在福尔马林固定的组织和细胞系。利用噬菌体抗体库技术，筛选出一个多样性为8 × 109的抗完全糖基化重组CD 133的人源性单链抗体库。针对去糖基化的CD 133进行计数器筛选以选择优先识别糖基化非依赖性表位的克隆。通过流式细胞术分析前导scFv，并将其克隆到兔免疫球蛋白支架中用于免疫组织化学（IHC）。发现命名为HA 10的抗体以高亲和力结合CD 133的肽骨架上的糖基化非依赖性表位。作为流式细胞术的试剂，HA 10比通常使用的市售抗体更准确地检测CD 133。用HA 10进行的IHC分析记录了健康前列腺切片中基底细胞和腔细胞的染色。在腺癌切片中以极低频率观察到腔细胞的弱染色。LuCaP患者来源的异种移植组织微阵列的检查发现，只有三个LuCaP模型对CD 133呈阳性。三种⑶ 133 pos LuCaP模型均源自具有神经内分泌分化的非AR驱动的转移性前列腺癌。随后对第二代抗雄激素治疗失败的患者的肝活检进行询问，发现高水平的CD 133染色。然而，该患者最初的经尿道前列腺切除术缺乏CD 133。我们已经开发了一种新的抗体，能够通过IHC和流式细胞术检测CD 133。使用HA 10作为IHC试剂，我们发现CD 133是健康前列腺和腺癌切片中非常罕见的细胞类型的标志物。我们的初步研究还表明，CD 133和非AR驱动的前列腺癌与神经内分泌分化之间可能存在关联。
The transmembrane glycoprotein CD133 is believed to be a marker of adult prostate stem cells and cancer stem/initiating cells. Investigating the role of CD133 in the normal biology of the prostate and in cancer is complicated by the lack of a sensitive and accurate antibody for its detection. Here, we describe the characterization of a unique antibody identified using human antibody phage display that can recognize CD133 in both formalin-fixed tissues and cell lines. A human single-chain variable fragment (scFv) antibody phage display library possessing a diversity of 8 × 109 was screened against fully glycosylated recombinant CD133. A counter screen was performed against deglycosylated CD133 to select for clones that preferentially recognized a glycosylation-independent epitope. The lead scFv was analyzed by flow cytometry and cloned into a rabbit immunoglobulin scaffold for immunohistochemistry (IHC). The antibody designated HA10 was found to bind a glycosylation-independent epitope on the peptide backbone of CD133 with high affinity. As a reagent for flow cytometry, HA10 detected CD133 more accurately than a commonly used commercially available antibody. IHC analysis with HA10 documented the staining of basal cells and luminal cells in healthy prostate sections. Weak staining of luminal cells was observed in adenocarcinoma sections at a very low frequency. Examination of a LuCaP patient-derived xenograft tissue microarray found that only three of the LuCaP models were positive for CD133. The three CD133pos LuCaP models all originated from non-AR driven metastatic prostate cancer with neuroendocrine differentiation. Subsequent interrogation of liver biopsies from a patient who failed second-generation anti-androgen therapy found high levels of CD133 staining. The original transurethral resection of the prostate from that patient was, however, absent of CD133. We have developed a novel antibody that was able to detect CD133 by both IHC and flow cytometry. Using HA10 as an IHC reagent, we found that CD133 is a marker for a very rare cell type in both healthy prostate and adenocarcinoma sections. Our preliminary investigation also suggests that there may be an association between CD133 and non-AR driven prostate cancer with neuroendocrine differentiation.
登录
查看更多内容
影响因子：
11.2
作者：
Vander Griend DJ;Karthaus WL;Dalrymple S;Meeker A;DeMarzo AM;Isaacs JT
通讯作者：
Isaacs JT
影响因子：
3.7
作者：
Thamm K;Graupner S;Werner C;Huttner WB;Corbeil D
通讯作者：
Corbeil D
影响因子：
82.9
作者：
Beltran H;Prandi D;Mosquera JM;Benelli M;Puca L;Cyrta J;Marotz C;Giannopoulou E;Chakravarthi BV;Varambally S;Tomlins SA;Nanus DM;Tagawa ST;Van Allen EM;Elemento O;Sboner A;Garraway LA;Rubin MA;Demichelis F
通讯作者：
Demichelis F
影响因子：
3.7
作者：
Barrantes-Freer A;Renovanz M;Eich M;Braukmann A;Sprang B;Spirin P;Pardo LA;Giese A;Kim EL
通讯作者：
Kim EL
影响因子：
2.8
作者：
Isaacs, John T.
通讯作者：
Isaacs, John T.