Key Laboratory of Pathogenic Fungi and Mycotoxins of Fujian Province, Key Laboratory of Biopesticide and Chemical Biology of Education Ministry, and School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China.
Fuzhou Maixin Biotech. Co., Ltd, Fuzhou, 350100, China.
BMC Biotechnol. 2018 May 4;18(1):25. doi: 10.1186/s12896-018-0436-z.
Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker.
The synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG, and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 10 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively.
A sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis.
人嗜铬粒蛋白 A（CgA）是一种约 49 kDa 的分泌蛋白，主要来自神经内分泌细胞和内分泌细胞。CgA 值在肿瘤诊断中具有重要价值，并且作为生物标志物在肿瘤的预后和预测方面具有潜在作用。
成功克隆了 CgA 编码区的合成基因，并在原核系统中表达为融合蛋白 CgA-His。然后将纯化的 CgA-His 蛋白与 QuickAntibody-Mouse5W 佐剂混合，注入小鼠体内。CgA-His 蛋白也被用作包被抗原来确定抗血清效价。通过筛选，获得了一株可产生抗 CgA 单克隆抗体（mAb）的稳定细胞系，命名为 4E5。4E5 mAb 的亚型为 IgG，染色体数为 102±4。从腹水液中纯化出抗 CgA mAb，亲和常数达到 9.23×10^L/mol。此外，还通过 ELISA、western blot 和免疫组织化学法确定了 mAb 的特异性。结果表明，mAb 4E5 能够特异性和灵敏地检测嗜铬粒蛋白 A。
本研究成功开发了一种快速生产用于免疫组织化学诊断的抗 CgA mAb 的敏感可靠方法，为制备 mAb 并在免疫组织化学诊断中实施提供了明确的指导。
