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目的制备特异性抗人LOC339524的单克隆抗体(m Ab)并进行应用。方法将LOC339524蛋白的非糖基化抗原序列的编码基因进行三段重复表达,合成的基因克隆到质粒p ET28a上。采用大肠杆菌表达系统获得LOC339524重组蛋白,以此作为免疫原免疫BALB/c小鼠,获得m Ab。ELISA测定抗体特异性及效价。Western blot法鉴定LOC339524重组蛋白、免疫组织化学技术分别检测人心肌组织和H9C2细胞中LOC339524蛋白的表达,采用制备的抗体检测不同心脏病患者血清LOC339524水平。结果筛选出2株能稳定分泌抗人LOC339524蛋白m Ab的杂交瘤细胞株,5-D3和4-F8。其中5-D3效价高于4-F8,达2×106。Western blot法、免疫组织化学技术证明5-D3能特异性识别人和大鼠LOC339524蛋白,应用制备的抗体可成功检测不同心脏病患者血清LOC339524的水平。结论成功制备了特异性好、效价高的抗人LOC339524蛋白m Ab。
Objective To prepare and use monoclonal antibody (m Ab) specific against human LOC339524. Methods The gene coding for the non-glycosylated antigen sequence of LOC339524 protein was expressed in three segments repeatedly. The synthesized gene was cloned into plasmid pET28a. The E.coli expression system was used to obtain LOC339524 recombinant protein, which was used as an immunogen to immunize BALB / c mice to obtain m Ab. Antibody specificity and titer were determined by ELISA. The LOC339524 recombinant protein was identified by Western blot. The expression of LOC339524 protein was detected by immunohistochemistry in human cardiac muscle and H9C2 cells. The serum levels of LOC339524 in patients with different cardiac diseases were detected by using the prepared antibodies. Results Two hybridoma cell lines, 5-D3 and 4-F8, which can stably secrete anti-human LOC339524 protein m Ab were screened out. The 5-D3 titer was higher than 4-F8, reaching 2 × 106. Western blot and immunohistochemistry showed that 5-D3 could specifically recognize human and rat LOC339524 protein. The prepared antibody could successfully detect serum levels of LOC339524 in patients with different heart diseases. Conclusion The anti-human LOC339524 protein m Ab was successfully prepared with good specificity and high titer.