4 u00b0 c beads were washed three times with 0 1 bsa pbs transfected cell culture supernatants were incubated with beads for 1 night at 4 u00b0 c washed beads were then resuspended in sds page sample buffer for electrophoresis reduced and unreduced immunoprecipitates were subjected to electrophoresis in a 5 sds acrylamide gel sepharose columns were purchased from diamed paris france twenty microliters of a 2 5 suspension of e were incubated for 45 minutes at 37 u00b0 c with 50 u03bcl of supernatant of transfected cells agglutination was then assessed in columns after a 1 000g centrifugation for 10 minutes at room temperature ttfgc c4bp u03b1 anti gpa scfv c4bp u03b1 binding on erythrocytes was analysed by flow cytometry washed e were incubated for 1 hour at room temperature with transfected cells supernatant then washed three times with 1 bsa pbs human serum or 1 u03bcg of anti tetanus toxin fragment c roche indianapolis usa were added for 45 minutes at room temperature erythrocytes were washed twice and then 1 u03bcg of goat anti human ig h l biotinylated antibody southern biotechnology associates birmingham usa or anti mouse ig biotinylated antibody amersham biosciences buckinghamshire uk were added before 1 5 u03bcg of streptavidin r phycoerythrin conjugated antibody tebu bio burlingame usa conjugated detection.