50 ml and the mixture was then stirred at rt for 6 hours the mixture was concentrated to dryness and the crude product obtained was triturated with ether and the solid formed was filtered washed with ether and dried under vacuum overnight to give 4 4 benzylpiperazin 1yl propionic acid tfa salt hapten 3 10 5 72 as illustrated in fig4 to a solution of 4 4 benzylpiperazin 1 yl propionic acid tfa salt hapten 3 54 35 mg 0 15 mm in dmf 1 0 ml was added n n dicyclohexylcarbodiimide dcc 33 08 mg 0 16 mm and n hydroxysuccinimide 18 7 mg 0 16 mm and the mixture was stirred at room temperature overnight the dicyclohexylurea formed was removed by filtration and the solution was added drop wise to a solution of bsa 150 mg 2 3 u03bcmol in 50 mm sodium bicarbonate solution ph 8 5 10 ml the mixture was then stirred overnight at 4 u00b0 c the solution was then dialysed against 50 mm phosphate buffer ph 7 2 3 changes for 24 hours at 4 u00b0 c and freeze dried maldi results showed 6 03 molecules of hapten 3 had been conjugated to one molecule of bsa to a solution of 4 4 benzylpiperazin 1 yl propionic acid tfa salt hapten 3 73 55 mg 0 203 mmol in dmf 1 0 ml was added n n dicyclohexylcarbodiimide dcc 46 01 mg 0 223 mmol and n hydroxysuccinimide 25 66 mg 0 223 mmol and the mixture was stirred at room temperature overnight the dicyclohexylurea formed was removed by filtration and the solution was added drop wise to a solution of btg 150 mg 2 25 u03bcmol in 50 mm.