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目的 :制备兔抗小鼠脾酪氨酸激酶 (Syk)抗体 ,分析其在乳腺癌诊断中的价值。方法 :应用重组小鼠Syk蛋白 ,常规免疫雄性家兔制备抗Syk抗体。用免疫印迹法检测Syk抗体的纯度和特异性。应用免疫组化法 ,检测乳腺组织中Syk蛋白的表达。结果 :用重组小鼠Syk蛋白免疫家兔 6wk后 ,静脉血中抗Syk抗体效价为 1∶6 4 0 0 (A450 =1.0 3)。将Syk经SDS PAGE分离 ,可见 1条相对分子质量 (Mr)为 72 0 0 0的蛋白条带 ,与理论值相符。用抗Syk血清做免疫印迹证实 ,在Mr 为 72 0 0 0处有 1条明显的带 ,证明是抗Syk特异性抗体。乳腺组织切片经抗Syk抗体免疫组化染色 ,在正常乳腺组织细胞胞浆中可见棕黄色颗粒 ,而浸润性乳腺导管癌组织的细胞浆不着色。结论 :制备了高效价、特异性的兔抗Syk抗体。用该抗体检测证实 ,Syk在正常乳腺组织细胞中呈高表达 ,在乳腺癌组织细胞中表达缺失。有可能用于乳腺癌的临床诊断
Objective: To prepare rabbit anti-mouse spleen tyrosine kinase (Syk) antibody and analyze its value in the diagnosis of breast cancer. Methods: Recombinant mouse Syk protein was used to immunize male rabbits to prepare anti-Syk antibody. The purity and specificity of Syk antibody was measured by Western blotting. Immunohistochemistry was used to detect the expression of Syk protein in breast tissue. RESULTS: The anti-Syk antibody titer in venous blood was 1: 640 (A450 = 1.03) after immunized rabbits with recombinant mouse Syk protein for 6 weeks. Syk was separated by SDS PAGE, showing a relative molecular mass (Mr) of 72 0 0 0 protein bands, consistent with the theoretical value. Immunoblotting with anti-Syk serum confirmed that there was a distinct band at Mr 720, which proved to be an anti-Syk-specific antibody. The sections of mammary gland were stained by anti-Syk antibody immunohistochemically. Brown-yellow granules were seen in the cytoplasm of normal breast tissues, while the cytoplasm of invasive breast ductal carcinoma was not stained. Conclusion: High titer and specific rabbit anti-Syk antibody was prepared. Using this antibody test, Syk was highly expressed in normal breast tissue cells and was absent in breast cancer tissue cells. It is possible for the clinical diagnosis of breast cancer