rev: July 22, 1999

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Extraction Protocol for IBL Steroid Elisa Tests code cat#RDI-RE52xxx

1)  Material for Preparation of Tests:

      a)    Diethyl-ether

      b)   human serum free from steroid

      c)   1.5ml safe-lock test tubes (eppendorf)

      d)  vortexing device VF2

      e) centrifuge

      f) -80 DEG C freezer

      g)  Sonorex ultrasound bath

      h) pipets 40-2000ul

Extraction Procedure:

a) Mix 230ul serum with 1ml diethyl-ether

b) Vortex for 20 seconds and shake thoroughly for 15 minutes

c) Centrifuge for 5 minutes at >1500 g

d) Freeze-out upper organic phase at -80DEG C.

e) Repeat steps 1-4 and bring the extracts together

f) Evaoprate extracts down to 100ul (e.g. with nitrogen) in a water bath at 35 DEg C. Wash surface of the tube with 150ul of diethyl-ether, vortex shortly

g) Evaporate extracts down to about 20ul and add 75ul steroid free serum

h) Centrifuge for 3 minutes at  >1500 X g

i) Evaporate the remaining diethyl-ether

j) Place the tube for 12 minutes in an ultra sonic bath

k) Centrifuge for 3 minutes at /1500 X g

l) The clear liquid sample may be used as described in the assay procedure..

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