Determining Specificity of Fab Antibody against Mouse Serologically Detected Male Antigen

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.