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::  p r o t o c o l s  ::  p l a t e l e t - a s s o c i a t e d  Ig  e l u t i o n  ::


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OUTLINE

Platelet-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.

PROTOCOL

I. GLYCINE-HCl Elution
  1. 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
  2. Incubate at RT for 15 min.
  3. Centrifuge at 14000 rpm, 5min , RT or 10ƒC
  4. Neutralize with 1mol/L Tris-base (pH=9.0)
  5. Dialyze ON, 4ƒC, against PBS
  6. Centrifuge at 14000 rpm, 5min , RT or 10ƒC
II. ETHER Elution
  1. Resuspend the platelet pellet (10^8 Platelets/mL) in 400µl [PBS-EDTA 5mM-BSA 2%] 
  2. add 800µl of diethylether
  3. vigorously mix for 3 minutes 
  4. incubated afterwards at 37ƒC for 45 minutes in 1,5 ml plastic tube without additional shaking. 
  5. let the tube overnight at 4ƒC for ether evaporation 
  6. centrifuged at room temperature for 10 minutes at 5000g
SOLUTIONS
I.
  1. 0,1 mol/L glycine HCl (pH=2.5)
  2. 1mol/L Tris-base (pH=9.0)
  3. PBS
II.
  1. PBS-EDTA 5mM-BSA 2%
  2. diethylether extra-pure
ADDITIONAL INFO
If 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
  1. 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
  2. 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.
created by Andrei Musaji          ::          updated : 2004