This document describes the steps for a radioimmunoassay to measure steroid levels in plasma samples. The process involves: 1. Preparing plasma samples and adding labeled steroid. 2. Extracting steroids from the plasma using dichloromethane and column packing. 3. Performing column chromatography to separate the steroids. 4. Setting up the separated steroids and standards for radioimmunoassay to measure steroid levels. 5. Using dextran-coated charcoal to separate bound and unbound steroid for counting.

1. STEROIDSTEROID
RADIOIMMUNOASSAYRADIOIMMUNOASSAY
Steps :Steps :
1. PREPARATION OF PLASMA1. PREPARATION OF PLASMA
SAMPLES (Day 1)SAMPLES (Day 1)
2.2. EXTRACTION OF STEROIDS AND COLUMNEXTRACTION OF STEROIDS AND COLUMN
PACKING (Day 2PACKING (Day 2))
3.3. COLUMN CHROMATOGRAPHY (Day 2-3)COLUMN CHROMATOGRAPHY (Day 2-3)
4.4. RADIOIMMUNOASSAY (Day 4)RADIOIMMUNOASSAY (Day 4)
5.5. SEPARATION OF BOUND AND FREESEPARATION OF BOUND AND FREE
COUNTS (Day 5)COUNTS (Day 5) andand CALCULATIONSCALCULATIONS

2. PREPARATION OF PLASMAPREPARATION OF PLASMA
SAMPLESSAMPLES
Set up plasma samples in glass centrifuge tubes (pointedSet up plasma samples in glass centrifuge tubes (pointed
bottoms, 12 ml). The first and last tubes are always blanksbottoms, 12 ml). The first and last tubes are always blanks
and contain only distilled water (dH2O). The second tube isand contain only distilled water (dH2O). The second tube is
always aalways a standardstandard into which measured amounts of theinto which measured amounts of the
steroids being assayed are placed. This measures thesteroids being assayed are placed. This measures the
accuracy of the current assay as well as interassayaccuracy of the current assay as well as interassay
variation, when samples may be spread over more thanvariation, when samples may be spread over more than
one assay. When all plasma samples have been measured,one assay. When all plasma samples have been measured,
a small amount of labelled steroid (20 µl of each steroida small amount of labelled steroid (20 µl of each steroid
being tested=2,000cpm) is added to all tubes except thebeing tested=2,000cpm) is added to all tubes except the
blanks. Total cpm is measured at the end to determineblanks. Total cpm is measured at the end to determine
what percentage of the labelled steroid is recovered, and,what percentage of the labelled steroid is recovered, and,
assuming that the unknown steroid behaves the same, theassuming that the unknown steroid behaves the same, the
final dose of the unknown steroid is adjusted according tofinal dose of the unknown steroid is adjusted according to
thisthis recoveryrecovery value.value.

3. EXTRACTION OF STEROIDSEXTRACTION OF STEROIDS
AND COLUMN PACKING (Day 2AND COLUMN PACKING (Day 2))
 Add 5 mls distilled dichloromethaneAdd 5 mls distilled dichloromethane
to each sample.to each sample.
 Vortex each sample at a low speed,Vortex each sample at a low speed,
wearing protective gloves andwearing protective gloves and
clothing.clothing.
 Let tubes stand for at least 2 hours.Let tubes stand for at least 2 hours.
 Progesterone must be extracted byProgesterone must be extracted by
ethyl etherethyl ether

4. COLUMN CHROMATOGRAPHYCOLUMN CHROMATOGRAPHY
(Day 2-3)(Day 2-3)
Adding samples to columns.Adding samples to columns.
Using EP, add 0.5 ml of 10% ethyl acetateUsing EP, add 0.5 ml of 10% ethyl acetate
in iso-octane to each sample. Vortex firstin iso-octane to each sample. Vortex first
tube, and add contents to first columntube, and add contents to first column
with a disposable pipet. Add another 0.5with a disposable pipet. Add another 0.5
ml of 10% mix to test tube, voretx andml of 10% mix to test tube, voretx and
add to column (2nd time is to rinse).add to column (2nd time is to rinse).
Leave pipet stuck in column to mark yourLeave pipet stuck in column to mark your
place and to avoid adding two samples toplace and to avoid adding two samples to
the same column. Proceed to the nextthe same column. Proceed to the next
sample. When all have been placed onsample. When all have been placed on
columns remove and discard the pipetcolumns remove and discard the pipet

5. Attach hoses to columns and turn on nitrogen.Attach hoses to columns and turn on nitrogen.
Regulate the pressure so that none drips faster than 1 dripRegulate the pressure so that none drips faster than 1 drip
every 6 seconds. When the solvent reaches the top of theevery 6 seconds. When the solvent reaches the top of the
celite turn off that column and remove the hose. Do not drycelite turn off that column and remove the hose. Do not dry
out. Discard this eluate using methods approved by yourout. Discard this eluate using methods approved by your
institution. Columns may now be left overnight at any pointinstitution. Columns may now be left overnight at any point
after steroids have been added. To do this add theafter steroids have been added. To do this add the
appropriate ethyl acetate/iso-octane mix, attach hoses, turnappropriate ethyl acetate/iso-octane mix, attach hoses, turn
on nitrogen to get things started, then turn off nitrogen aton nitrogen to get things started, then turn off nitrogen at
tank and allow a slow drip overnight. They will not run drytank and allow a slow drip overnight. They will not run dry
provided nitrogen was turned off at tank. When running theprovided nitrogen was turned off at tank. When running the
following fractions, collect only those wanted, but allfollowing fractions, collect only those wanted, but all
fractions must be run unless noted below. Collect intofractions must be run unless noted below. Collect into
13x100 mm test tubes and place in a rack for evaporation.13x100 mm test tubes and place in a rack for evaporation.

6.  Add 4 mls pure iso-octane and blow downAdd 4 mls pure iso-octane and blow down
under regulated pressure as above. Theunder regulated pressure as above. The
eluate iseluate is PP. However, if P will be collected,. However, if P will be collected,
use 4 mls of 2% ethyl acetate in iso-use 4 mls of 2% ethyl acetate in iso-
octane instead of pure iso-octane.octane instead of pure iso-octane.
 Add 4.5 mls 10% ethyl acetate in iso-Add 4.5 mls 10% ethyl acetate in iso-
octane as above. The eluate isoctane as above. The eluate is DHTDHT..
 Add 4.5 mls 20% ethyl acetate in iso-Add 4.5 mls 20% ethyl acetate in iso-
octane. The eluate isoctane. The eluate is TT..
 Add 4.5 mls 40% ethyl acetate in iso-Add 4.5 mls 40% ethyl acetate in iso-
octane. The eluate isoctane. The eluate is E2E2. This step is. This step is
optional and done only when assaying foroptional and done only when assaying for
E2.E2.
 Add 4.0 mls 50% ethyl acetate in iso-Add 4.0 mls 50% ethyl acetate in iso-
octane (4.5 mls if you skipped the 40%octane (4.5 mls if you skipped the 40%
fraction). The eluate isfraction). The eluate is BB..

7.  Dry purified extracts under nitrogenDry purified extracts under nitrogen
evaporator in a 40oC water bathevaporator in a 40oC water bath
until totally evaporated. Add 550 µluntil totally evaporated. Add 550 µl
buffer (PBSG) to each tube for allbuffer (PBSG) to each tube for all
except B. Add 1 ml buffer to B tubes.except B. Add 1 ml buffer to B tubes.
Buffer can be added using aBuffer can be added using a
combination of tips on the ER or withcombination of tips on the ER or with
a variable pipet. Vortex, cover (witha variable pipet. Vortex, cover (with
foil or parafilm), and refrigeratefoil or parafilm), and refrigerate
overnight. Note: You may place rackovernight. Note: You may place rack
on shaker and shake for 45 minuteson shaker and shake for 45 minutes
and proceed to set up curves.and proceed to set up curves.

8. RADIOIMMUNOASSAY (Day 4)RADIOIMMUNOASSAY (Day 4)
Setting up the samples.Setting up the samples.
Vortex each tube before partitioning. UsingVortex each tube before partitioning. Using
an EP, pipet 200 µl of each sample intoan EP, pipet 200 µl of each sample into
duplicate assay tubes. Either simultaneouslyduplicate assay tubes. Either simultaneously
or following, pipet 100 µl into a scintillationor following, pipet 100 µl into a scintillation
vial for the recovery. Scintillant will then bevial for the recovery. Scintillant will then be
added to these vials. Cap and vortexadded to these vials. Cap and vortex

9. Setting up the standard curve:Setting up the standard curve:
The standard curve will be used toThe standard curve will be used to
determine the dose of steroid in thedetermine the dose of steroid in the
unknown samples. The first three tubes areunknown samples. The first three tubes are
B1-B3. B1 measures total cpm, B2 non-B1-B3. B1 measures total cpm, B2 non-
specific binding (or background), and B3specific binding (or background), and B3
maximum binding with the antiserum. Themaximum binding with the antiserum. The
remaining 9 pairs of tubes (S1-S9) generateremaining 9 pairs of tubes (S1-S9) generate
the curve.the curve.

10. SEPARATION OF BOUND ANDSEPARATION OF BOUND AND
FREE COUNTS Day 5FREE COUNTS Day 5
Dextran-coated charcoal will be addedDextran-coated charcoal will be added
to allto all
tubes (except B1) and allowed totubes (except B1) and allowed to
adsorb alladsorb all
unbound steroid. Tubes will beunbound steroid. Tubes will be
centrifuged,centrifuged,
and the supernatant will be decantedand the supernatant will be decanted
into 7into 7
ml scintillation vials for counting cpm.ml scintillation vials for counting cpm.