Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Bioconjug Chem. 2009 Aug 19;20(8):1474-81. doi: 10.1021/bc800421f. Epub 2009 Jul 31.
The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.
本工作展示了小双价工程抗体片段半胱氨酸二抗体（cys-diabodies）在生物修饰纳米颗粒（如量子点（Qdots））方面的应用，用于检测靶抗原。通过将肿瘤特异性 cys-diabodies 巯基特异性定向偶联到远离抗原结合位点的位置，合成了新型生物共轭量子点，称为免疫 Qdots（iQdots）。最初，通过胺巯基反应性连接物[N-ε-马来酰亚胺基羰基氧基]琥珀酰亚胺酯（EMCS）将还原的抗 HER2 cys-diabody 与氨基聚乙二醇 CdSe/ZnS Qdot 655 偶联，生成抗 HER2 iQdot 655。偶联物的光谱特征表明，其光谱是对称的，与未偶联的 Qdot 基本相同。通过流式细胞术在 HER2 阳性和阴性细胞上证实了抗 HER2 iQdot 655 的特异性受体结合活性。免疫荧光结果表明，Qdot 655 缀合物均匀地标记了细胞膜表面。此外，成功地将针对 HER2 以及前列腺干细胞抗原（PSCA）的 cys-diabodies 与氨基聚乙二醇 Qdot 800 偶联。所有这些 iQdots 均保留了未偶联的 Qdot 800 的光致发光特性，以及 cys-diabody 的抗原结合特异性，如流式细胞术所证实的那样。使用两种 iQdots，即抗 HER2 iQdot 655 和抗 PSCA iQdot 800，能够同时检测培养中的 LNCaP/PSCA 前列腺癌细胞（表达 PSCA 和 HER2）上的两种肿瘤抗原。因此，这些 iQdots 可能是用于敏感、多重检测肿瘤细胞表面标志物的光学探针。本巯基特异性偶联方法展示了一种通用方法，用于将 cys-diabodies 特异性定向偶联到各种纳米颗粒上，而不会干扰抗原结合位点，并与完整抗体相比保持较小的尺寸。
