Division of Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, PR China.
Med Sci Monit. 2012 May;18(5):BR167-73. doi: 10.12659/msm.882725.
ZNF32 has been predicted to be a zinc finger protein and is involved in cell differentiation and tumor development, but its precise function is unknown. Specific monoclonal antibodies (mAbs) have been widely used in research and clinical diagnosis and treatments. Therefore, we established an anti-ZNF32 mAb to characterize this protein's function.
MATERIAL/METHODS: Peptide⁴⁹⁻⁶³, a specific small peptide of ZNF32, was chosen and the synthetic keyhole limpet hemocyanin (KLH)-peptide⁴⁹⁻⁶³ was used as an antigen to immunize mice. A mAb against peptide⁴⁹⁻⁶³ was generated by hybridoma technology, and hybridoma cells were screened by limiting dilution. The isoform of mAb-pZNF32-8D9 was identified by double agar diffusion. The sensitivity and specificity of the mAb and expressed levels of ZNF32 in various cells and tissues were identified by enzyme-linked immunosorbent assay (ELISA), immunocytochemistry, immunohistochemistry, and Western blotting.
A stable anti-pZNF32-8D9 hybridoma secreting the anti-peptide49-63 mAb was established and the clone positive to the peptide⁴⁹⁻⁶³ in supernatant was 92% in ELISA. The mAb-pZNF32-8D9 is an immunoglobulin-1 that can be used for detecting the ZNF32 protein by immunocytochemistry, immunohistochemistry, and Western blotting and is highly sensitive and specific. We also found ZNF32 expressed at high levels in Jurkat and pulmonary squamous carcinoma cells, but it was not expressed in squamous epidermis cells.
mAb-pZNF32-8D9 can be used for the identification and expression of ZNF32. It might also provide a new tool for diagnostics or therapy for ZNF32-related diseases.
ZNF32 被预测为一种锌指蛋白，参与细胞分化和肿瘤发生，但确切功能尚不清楚。特异性单克隆抗体（mAb）已广泛应用于研究和临床诊断治疗。因此，我们建立了抗 ZNF32 mAb 以研究该蛋白的功能。
材料/方法：选择 ZNF32 的特定小肽肽 49-63，并用合成的血蓝蛋白（KLH）-肽 49-63 作为抗原免疫小鼠。利用杂交瘤技术制备针对肽 49-63 的 mAb，通过有限稀释筛选杂交瘤细胞。通过双琼脂扩散鉴定 mAb-pZNF32-8D9 的同种型。通过酶联免疫吸附试验（ELISA）、免疫细胞化学、免疫组织化学和 Western blot 鉴定 mAb 的灵敏度和特异性以及不同细胞和组织中 ZNF32 的表达水平。
建立了稳定分泌抗肽 49-63 mAb 的抗-pZNF32-8D9 杂交瘤细胞株，ELISA 检测上清液中对肽 49-63 呈阳性的克隆为 92%。mAb-pZNF32-8D9 为免疫球蛋白-1，可通过免疫细胞化学、免疫组织化学和 Western blot 检测 ZNF32 蛋白，灵敏度和特异性高。我们还发现 ZNF32 在 Jurkat 和肺鳞癌细胞中高表达，但在鳞状表皮细胞中不表达。
mAb-pZNF32-8D9 可用于鉴定和表达 ZNF32，也可能为 ZNF32 相关疾病的诊断或治疗提供新的工具。
