摘要
目的 制备OVA6 6蛋白和其抗体,探讨其检测肿瘤的应用价值。方法 应用原核表达技术和Ni2 + 亲和层析法,制备纯化的OVA6 6蛋白,经SDS- PAGE电泳鉴定,将纯化蛋白加佐剂免疫家兔,获得抗OVA6 6蛋白抗血清,并应用WesternBlot测定其滴度和特异性。利用OVA6 6蛋白和其抗体分别建立ELISA和免疫组化法,测定正常人和肿瘤患者的血清OVA6 6抗体水平和肿瘤组织标本中OVA6 6蛋白表达量。结果 获得的高纯度OVA6 6蛋白和高滴度、特异性抗OVA6 6抗血清。ELISA检测显示,正常人和肿瘤患者血清抗体水平存在显著性差异(P <0 .0 0 1)。正常人组OD均值为0 . 2 0±0 0 5 ,肿瘤患者组OD均值为0 . 36±0. 12 ,肿瘤患者中阳性率为4 0 % (46 / 116 )。免疫组化法结果表明,肿瘤组织标本阳性率达81% (34/ 4 2 ) ,而正常组织均为阴性。结论 应用OVA6 6蛋白和其抗血清建立的检测方法,为临床肿瘤辅助诊断的应用奠定基础。
Objective To prepare OVA66 protein and anti-OVA66 antibody serum, and study applicable value of detecting clinical tumor. Methods Prokarytic expressing OVA66 protein was purified through Ni 2+ affinity chromatography column, The OVA66 protein was injected into rabbits to make anti-OVE66 serum, the protein and its antibody titer were detected by SDS-PAGE and Western Blot, respectively. ELISA and immunohistochemical technique were established to analyze anti-OVA66 antibody level in normal and tumor patient's serum, and the OVA66 protein expression in the tumor tissues, which to compare normal and tumor tissue expression.Results The purified OVA66 protein and high titer anti-OVA66 serum were obtained. ELISA results showed OD mean's of normal serum group was 0.20±0.05, and tumor patient's serum group was 0.36±0.12, the difference was significant, positive rate was 40%(46/116) of tumor serum. Immunohistochemical results showed positive rate was 81%(34/42) of tumor tissue, whereas it was negative of normal tissues. Conclusion The purified protein and its antibody serum were obtained, which were used to establish ELISA and immunohistochemical method that could develop novel application for diagnosis of clinical tumors.
出处
《中国实验诊断学》
2005年第2期198-200,共3页
Chinese Journal of Laboratory Diagnosis
基金
国家自然科学基金项目(No.30371598)
上海市高等学校科学技术发展基金项目(No.03BX20)
上海浦东新区社发局医学科研项目(NO.PW2003-A23)。
