The lymphocytes were collected from the EDTA samples of CLL patients by centrifugation and the supernatants removed. The cells were washed 3 times and then resuspended in tampon phosphate (PBS). Lysis of cells by ultrasound was performed 4 times. The cells were centrifuged at 1500 × g for 10 minutes at a temperature within the range 2°C-8°C to remove other cellular debris. Alternatively, the cells could have been frozen at -20°C and warmed to room temperature for 3 hours. The micro titer plate wells were covered with 100 μl of the appropriate antibody (capture antibody) ‘at a rate of’1 μg/ml-10 μg/ml in the coating buffer. The plate was covered and incubated overnight at 4°C, and then washed 3 times in ELISA Wash Buffer. To each well 150 μl of blocking solution was added and incubated for 60 minutes at 37°C. The mixture was then washed 4 times in wash buffer. The samples were diluted with wash buffer (ELISA), and 100 μl of target antigen and appropriately diluted standards were added into the relevant wells. The mixture was then incubated for 90 minutes at 37°C and then carefully washed 3 times in wash buffer.