Plateletassociated Ig (PAIg) Elution

OUTLINEPlatelet-associated Ig (PAIg) could be eluted from the platelets. As a result of this procedure platelets get destroyed and the eluted Ig is formed of platelet surface-bound Ig and , mainly, of internal pools of Ig (from a-granules). That is why the caution must be taken when interpreting results. PROTOCOLI. GLYCINE-HCl Elution Resuspend the platelet pellet in 0,1 mol/L glycine HCl (pH=2.5) to a final concentration 2-10 x 10^8 Platelets/mL Incubate at RT for 15 min. Centrifuge at 14000 rpm, 5min , RT or 10ѓC Neutralize with 1mol/L Tris-base (pH=9.0) Dialyze ON, 4ѓC, against PBS Centrifuge at 14000 rpm, 5min , RT or 10ѓC II. ETHER Elution Resuspend the platelet pellet (10^8 Platelets/mL) in 400µl [PBS-EDTA 5mM-BSA 2%] add 800µl of diethylether vigorously mix for 3 minutes incubated afterwards at 37ѓC for 45 minutes in 1,5 ml plastic tube without additional shaking. let the tube overnight at 4ѓC for ether evaporation centrifuged at room temperature for 10 minutes at 5000g SOLUTIONSI. 0,1 mol/L glycine HCl (pH=2.5) 1mol/L Tris-base (pH=9.0) PBS II. PBS-EDTA 5mM-BSA 2% diethylether extra-pure ADDITIONAL INFOIf the fraction you are interested in is IgM don't freeze the sample. To avoid the contamination in the tube add NaN 3 to the final concentration of 0,05%REFERENCES Bowditch, R.D., Tani, P., Fong, K.C. and McMillan, R. (1996). Characterization of autoantigenic epitopes on platelet glycoprotein IIb/IIIa using random peptide libraries. Blood 88(12): 4579-84 Kosugi, S., Tomiyama, Y., Shiraga, M., Kashiwagi, H., Mizutani, H., Kanakura, Y., Kurata, Y. and Matsuzawa, Y. (1996). Platelet-associated anti-glycoprotein (GP) IIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura mainly recognize cation-dependent conformations: comparison with the epitopes of serum autoantibodies. Thromb Haemost 75(2): 339-45.