血清学检测的雄性特异性抗原Fab抗体的特异性鉴定

Determining Specificity of Fab Antibody against Mouse Serologically Detected Male Antigen

旨在研究血清学检测的雄性特异性抗原噬菌体Fab抗体的特异性。以从噬菌体Fab抗体库中筛选到具有较高雄性特异性结合活性的重组噬菌体克隆为基础,构建Fab抗体的可溶性表达噬质粒,并诱导和纯化该抗体片段,利用免疫荧光FITC/DAPI共染技术和ELISA分析其特异性。结果表明,该抗体在约49 ku处有条带出现。在Fab抗体和H-Y抗血清的细胞免疫荧光比较分析试验中发现,从雌、雄鼠脾细胞的阳性细胞数量和平均荧光强度的角度分析,Fab抗体的雄性特异性强于抗血清。石蜡切片免疫荧光定量分析显示,Fab抗体与雄鼠肝脏的结合活性明显高于对应雌鼠,差异极显著(t=20.73,P=0.002 3<0.01),而H-Y抗血清与雄鼠肝脏的结合活性略高于对应雌鼠,差异显著(t=7.11,P=0.019 2<0.05)。以C57BL/6雄鼠脾细胞、睾丸细胞作为抗原的ELISA分析显示,Fab抗体具有雄性特异性,但Fab抗体的OD值低于抗血清。结果提示,Fab抗体的雄性特异性高于H-Y抗血清,但其雄性特异性结合活性低于H-Y抗血清,Fab抗体在具备雄性特异性的同时,仍有少量雌性非特异性结合,Fab抗体的体外亲和力成熟可作为后续研究工作,以获得高亲和力、高雄性特异性的可溶性Fab抗体分子。

To study specificity of Fab antibody against mouse serologically detected male antigen,based on recombinant phage clone with male specific activity screened from phage Fab antibody library,expression plasmid of soluble Fab antibody was constructed,antibody fragment was induced and purified,the antibody specificity was determined by immunofluorescence staining with FITC/DAPI and ELISA.The results showed that an about 49 ku band appeared in the SDS-PAGE,comparison of cell immunofluorescence staining of Fab antibody and H-Y antiserum showed that male specificity and binding activity of Fab antibody was higher than that of antiserum,based on number of positive cells and mean fluorescence intensity of male or female spleen cells.The quantitative analysis of immunofluorescence staining of paraffin sections showed that binding activity of Fab antibody against male mouse liver was obviously higher than that of female mouse liver,there was high significant difference between them（t=20.73,P=0.002 30.01）,binding activity of H-Y antiserum against male mouse liver was slightly higher than that of female mouse,there was significant difference between them（t=7.11,P=0.019 20.05）.The soluble Fab antibody had male specific activity in the ELISA with male mouse spleen and testicular cells as antigen,but OD values of Fab antibody were lower than that of antiserum.The results suggest that male specificity of Fab antibody is higher than that of H-Y antiserum,but its binding activity with male specificity was lower than that of H-Y antiserum,Fab antibody with male specificity had non-specific binding with a little female tissue or cells,affinity maturation of Fab antibody in vitro would be used as follow-up study,to screen soluble Fab antibody with high affinity and specificity against mouse serologically detected male antigen.