A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis
A novel VHH nanobody against the active site (the CA domain) of tumor-associated, carbonic anhydrase isoform IX and its usefulness for cancer diagnosis
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DOI：
10.1007/s10529-013-1340-1
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发表时间：
2014-01-01
影响因子：
2.7
通讯作者：
Gargari, Seyed Latif Mousavi
中科院分区：
文献类型：
--
作者：
Araste, Fatemeh;Ebrahimizadeh, Walead;Gargari, Seyed Latif Mousavi
碳酸酐酶IX（CAIX）的表达在肿瘤细胞中的缺氧条件下显著增加。CAIX活性由位于酶的胞外部分上的催化结构域（CA）执行。CAIX酶活性的中和降低了肿瘤细胞的恶性程度和存活率。为了抑制酶活性，使用噬菌体展示技术开发针对CAIX的CA结构域的VHH纳米抗体。用重组CAIX免疫骆驼后，通过淋巴细胞cDNA上的巢式PCR分离VHH片段。通过ELISA测试分离的纳米抗体的结合亲和力。具有最高结合亲和力的克隆（K24）以可溶形式表达。K24纳米抗体的亲和力被确定为约为100。2.3 x 10（-5）。K24纳米抗体以高选择性和特异性识别HeLa细胞系中表达的CAIX。因此，这些发现对于癌症的诊断和治疗是有用的。
Expression of carbonic anhydrase IX (CAIX) significantly increases under hypoxic conditions in tumor cells. CAIX activity is executed by the catalytic domain (CA) located on the extracellular part of the enzyme. Neutralization of CAIX enzymatic activity reduces malignancy and survival of tumor cells. To inhibit the enzymatic activity, a VHH nanobody was developed against the CA domain of CAIX using phage display technology. Following immunization of a camel with the recombinant CAIX, VHH fragments were isolated by nested PCR on lymphocyte cDNA. Binding affinity of isolated nanobodies was tested by ELISA. A clone (K24) with the highest binding affinity was expressed in a soluble form. Affinity of K24 nanobody was determined to be approx. 2.3 x 10(-5). K24 nanobody recognized the expressed CAIX in the HeLa cell lines with high selectivity and specificity. These findings thus have usefulness for the diagnosis and treatment of cancers.