多聚抗原肽免疫磁珠在制备单表位抗体中的应用

Application of multiple antigen peptide immunomagnetic beads in single-epitope antibody preparation

目的:建立用多聚抗原肽(multiple antigen peptides,MAP)包被磁珠制备单表位抗体的方法。方法:通过Fmoc法固相化学合成UreB的8分支单表位MAP,将其作为免疫原免疫小鼠,获得多克隆抗血清。将MAP以共价偶联的方式包被磁珠制备免疫磁珠(immunomagnetic beads,IB),通过IB从多克隆抗血清中纯化单表位抗体,荧光偏振(fluorescence polarization,FP)法鉴定抗体的特异性,SDS-PAGE鉴定抗体纯度,紫外分光光度法测定其回收率。结果:合成的MAP具有较强的免疫原性,免疫小鼠后得到的抗体滴度高达1∶12800。MAP制备免疫磁珠的最佳包被浓度为100mg/L,包被效率最高可达79%。应用MAP免疫磁珠从抗血清中纯化得到的单表位抗体,经鉴定与其他抗原表位无反应性,其纯度为95%,抗体的回收率为5.8%。结论:MAP包被的磁珠可快速分离纯化出针对某一抗原表位的特异性抗体,其性质类似单克隆抗体。这一方案在快速制备少量高特异性抗体中具有广阔的应用前景。

AIM： To prepare specific single-epitope antibody using immunomagnetic beads coated with multiple antigen peptides（MAP）. METHODS： Eight-branch MAP is synthesized by Fmoc SPPS. After immunizing the BALB/c mice with the MAP, polyclonal antiserum was obtained. Then, the immunomagnetic beads （IB） were prepared by coating with the MAP in covalent bond form and were used to purify the specific single-epitope antibody from the antiserum of immunized animals, RESULTS： The titer of antiserum was 1： 12 800, which showed the strong immunogenicity of MAP. The best coating concentration of MAP was 100 mg/L and the max coating efficiency was 79%. After purifying specific antibody from polyclonal antiserum using the IB, the obtained antibodies only interacted with one epitope rather than other epitopes. The purity of the antibody reached 9.5% and the mean recovery rate was .5.8%. CONCLUSION： The beads coated with MAP can be used to quickly isolate specific single-epitope antibody which is similar to mono- clonal antibody. This method provide a convenient shortcut in preparing small amount of antibody with high specificity.