is incubated in the wells for 2 hours 4 serum is pre treated incubated with a stabilizing solution 0 010m sodium borate using a 1 5 sample dilution at 4 u00b0 c for 4 12 hours this solution is then diluted 1 5 with 0 1 hsa 5 monoclonal antibody is diluted with 0 1 hsa at a dilution of approximately 1 o d 280 u00d7 10 000 6 to the washed pbs wells are added in turn 50 u03bcl of serum 1 25 see 4 above and 50 u03bcl of diluted antibody see 5 above 7 the plate is covered with a plastic sheet and incubated with gentle shaking at 4 u00b0 c for 12 18 hours 8 after washing the plate with pbs 100 u03bcl of goat anti mouse igm u03bc chain specific phosphatase labeled 1 4 000 10 000 is added and the plate is incubated at 4 u00b0 c with gentle shaking for 3 4 hours 9 to the washed wells are added 100 u03bcl of substrate 15 mg of paranitrophenyl phosphate in 12 ml of carbonate bicarbonate buffer ph 10 3 10 color is allowed to develop at room temperature 20 u00b0 22 u00b0 c color intensity is read at 405 nm in a dynatech automatic plate reader the buoyant density of the hca from ascites fluid and the kle 1 cell line was determined by gradient centrifugation in cesium trifluoroacetate according to methods known in the art samples of hca were centrifuged in 47 cesium trifluoroacetate at 130 000 u00d7 g for 72 h in a beckmann t 1250 rotor fractions of 1 ml were collected the density of each sample determined.