Farahi Lia, Ghaemimanesh Fatemeh, Milani Saeideh, Razavi Seyed Mohsen, Bayat Ali Ahmad, Rabbani Hodjattallah, Akhondi Mohammad Mehdi
Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran.
Clinic of Hematology and Oncology, Firoozgar Hospital, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Iran J Biotechnol. 2019 Jan 11;17(1):e2277. doi: 10.21859/ijb.2277. eCollection 2019 Jan.
The unique expression of fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL) has been previously reported. Detecting FMOD in CLL patients using specific anti-FMOD mAbs might provide a promising method in detection, monitoring, and prognosis of CLL.
In this study, we aimed for producing specific antibodies against FMOD to facilitate further cohort study of CLL, thus addressing FMOD as a potential target of detection.
Human FMOD gene (1087 bp) was extracted from genome of the CLL patients, and was cloned into the expression vector of pET-22b (+). The recombinant FMOD protein (rFMOD) was expressed in Escherichia coli. The purified rFMOD protein was used as an immunogen in rabbit and mice. Hybridoma technology was used to develop the monoclonal antibodies (mAbs). Polyclonal antibody (pAb) was purified from the rabbit sera using affinity column. The reactivity of anti-FMOD antibodies was assessed in ELISA, immunocytochemistry (ICC) and Western blot.
ICC results showed that the anti-FMOD antibodies specifically detected FMOD in CLL PBMCs and cell lines. The developed anti-FMOD pAb detected FMOD in CLL lysates, compared to healthy PBMCs, in Western blot and ELISA.
The developed anti-FMOD mAbs, and pAb specifically detect FMOD in CLL samples and might be used as research tools for further investigations in CLL.
先前已有报道称纤维调节蛋白（FMOD）在慢性淋巴细胞白血病（CLL）患者中存在独特表达。使用特异性抗FMOD单克隆抗体检测CLL患者体内的FMOD，可能为CLL的检测、监测及预后评估提供一种有前景的方法。
在本研究中，我们旨在制备针对FMOD的特异性抗体，以促进对CLL的进一步队列研究，从而将FMOD作为一种潜在的检测靶点。
从CLL患者基因组中提取人FMOD基因（1087 bp），并将其克隆至pET-22b（+）表达载体中。重组FMOD蛋白（rFMOD）在大肠杆菌中表达。纯化后的rFMOD蛋白用作兔和小鼠的免疫原。采用杂交瘤技术制备单克隆抗体（mAbs）。使用亲和柱从兔血清中纯化多克隆抗体（pAb）。通过酶联免疫吸附测定（ELISA）、免疫细胞化学（ICC）和蛋白质免疫印迹法评估抗FMOD抗体的反应性。
ICC结果显示，抗FMOD抗体可特异性检测CLL患者外周血单个核细胞（PBMCs）和细胞系中的FMOD。在蛋白质免疫印迹法和ELISA中，与健康PBMCs相比，所制备的抗FMOD pAb可检测到CLL裂解物中的FMOD。
所制备的抗FMOD mAbs和pAb可特异性检测CLL样本中的FMOD，可能用作进一步研究CLL的研究工具。
