摘要
目的:利用小鼠IgG抗体Fc片段高特异、高亲和寡核苷酸配基,构建实时定量免疫PCR检测方法,提高抗体检测的灵敏度。方法:用SELEX技术从随机寡核苷酸文库中筛选抗体Fc片段特异寡核苷酸配基,设计合成信标序列,通过不对称PCR法,制备IgG Fc片段的核酸信标配基分子;32P标记核酸信标配基,采用琼脂糖凝胶阻滞双显色法鉴定核酸信标配基与IgG Fc片段结合的亲和力和特异性;制备IgG Fc特异性寡核苷酸信标配基-抗体复合检测分子,构建小鼠IgG Fc片段特异核酸信标配基介导的实时定量免疫PCR检测方法。结果:制备了IgG Fc片段的核酸信标配基分子;凝胶阻滞放射自显影和考马斯亮蓝二次染色结果显示该核酸信标配基分子与IgG Fc片段具有高度亲和力和活性,而且只与非变性IgG结合,与变性IgG不结合;IgG Fc片段的特异核酸信标配基与IgG结合形成复合检测分子,有效完成了信号传递和实时定量PCR信号放大过程。结论:初步建立了一种全新的核酸信标配基介导的免疫PCR检测方法,可有效提高现有IgG类抗体免疫检测的灵敏度和特异性。
Objective： To construct realtime quantitative immunoPCR detection methods by using high specific and affinity oligonucleotide ligands against Fc fragment of mouse IgG for improving the antibody＇s detection sensi tivity. Methods： The oligonucleotide ligands against the Fc fragment of mouse IgG were selected by SELEX, the dsDNA beacon sequences were designed and synthesised. Then, the beaconligands were prepared by asymmetric PCR method. The beaconligands were labeled by 32pdATP, the binding affinity and specific of beaconligands with the Fc fragment of IgG were analysed by EMSA method. The composite detection molecule with beacon li gandsIgG was prepared and the oligonucleotide ligands mediated realtime quantitative immunoPCR detection methods was established. Results： The beacon ligands for Fc fragment of mouse IgG were prepared successfully. The EMSA analysis showed that the beaconligands bound to Fc fragment of IgG with high binding affinity, and only bound to the active IgG not to denatured IgG. The beaconligands bound with IgG for accomplishing effective ly signaling transmission and realtime PCR amplification. Conclusion： A new beaconligands mediated immu noPCR detection method was established initially and that can improve effectively the sensitivity and specificity of immuno detection of IgG.
出处
《生物技术通讯》
CAS
2012年第2期215-219,共5页
Letters in Biotechnology
基金
国家自然科学基金(30271219
30960104
81060180)
国家中小企业创新基金(06C26226200591)
甘肃省重大研发项目(GK SOU
GKS031030)
甘肃省重点能力建设实验室和甘肃省科技厅人才梯队建设项目
