Hemmati Azam, Hassannia Hadi, Milani Saeideh, Hadavi Reza, Ghaemimanesh Fatemeh, Rabbani Hodjattallah
Antibody-Antigen Engineering Department, Monoclonal Antibody Research Center, Avicenna Research Institute , ACECR, Tehran, Iran .
Monoclon Antib Immunodiagn Immunother. 2018 Feb;37(1):38-44. doi: 10.1089/mab.2017.0059.
Receptor tyrosine kinase ROR1 has been introduced as an interesting prognostic cancer marker in histopathology. The aim of this study was to produce a polyclonal antibody (PAb) against recombinant human ROR1 protein to be used as a tool for investigation of ROR1 expression in human cancer tissue blocks. The extracellular part of human ROR1 recombinant protein was expressed using pET-28b(+) plasmid in Escherichia coli Bl21(DE3) host. The recombinant ROR1, as a candidate immunogen, was purified and injected to a New Zealand rabbit. Followed by raising the titration of antibody, polyclonal anti-ROR1 antibody was purified through affinity chromatography column. After determining the purity of PAb anti-ROR1, its specific reactivity was assessed through various assessments. Flow cytometry analysis showed that PAb anti-ROR1 specifically recognizes ROR1 molecule in a number of positive and negative cell lines. Results obtained from detection of ROR1 in paraffin-embedded breast adenocarcinoma tissue blocks (n = 11) also demonstrated that PAb anti-ROR1 can effectively be used in immunohistochemistry. In conclusion, the developed anti-ROR1 PAb can be used as a tool for determining the prognostic value of ROR1 in histopathology of cancer tissues.
受体酪氨酸激酶ROR1已被作为组织病理学中一种有趣的癌症预后标志物引入。本研究的目的是制备一种针对重组人ROR1蛋白的多克隆抗体（PAb），用作研究人癌组织块中ROR1表达的工具。使用pET-28b(+)质粒在大肠杆菌BL21(DE3)宿主中表达人ROR1重组蛋白的细胞外部分。将重组ROR1作为候选免疫原进行纯化，并注射到一只新西兰兔体内。在提高抗体滴度后，通过亲和层析柱纯化多克隆抗ROR1抗体。在确定抗ROR1 PAb的纯度后，通过各种评估来评估其特异性反应性。流式细胞术分析表明，抗ROR1 PAb能特异性识别多种阳性和阴性细胞系中的ROR1分子。在石蜡包埋的乳腺腺癌组织块（n = 11）中检测ROR1的结果也表明，抗ROR1 PAb可有效地用于免疫组织化学。总之，所制备的抗ROR1 PAb可作为一种工具，用于确定ROR1在癌症组织病理学中的预后价值。
