Laboratory Protocols

Add 5 mls distilled dichloromethane to each sample. Vortex each sample at a low speed, wearing protective gloves and clothing. Let tubes stand for at least 2 hours. Progesterone must be extracted with ethyl ether - see Appendix V.

Packing Celite Columns. Celite (diatomaceous earth) should be heated at 1000oF for 24 hours prior to use. Range must be 950-1100oF, so oven gauge must be accurate. Remove from oven (with great care!) and allow to cool for about 45 minutes prior to weighing. Cooling can occur while dichloromethane is being added to samples.

Water trap. Mix 6 gms celite with 2 mls dH2O using mortar and pestle. Mix well and then mix some more. Place a #2 glass bead in the bottom of a 5 ml disposable pipet. Put a rubber band around the pipet to hold it in the rack. Position band around the 1.5 mark. Shake celite mix into the column, using a funnel, to about the 3.5 mark. Using a glass rod, pack firmly to the 4.5 mark. Generally, the celite mix packs to about 1/3 of the loose volume. Pack all water traps. Cover and save any leftover celite mix in case columns get broken during next phase and have to be redone.

Glycol phase. Mix propylene glycol (1,2 propandediol) and ethylene glycol in equal amounts. Add 3 mls of glycol mix to 6 gms celite and mix with a mortar and pestle until homogeneous. Add mix to the 3 mark on the column and pack down to 4. Add again to the 2.5 mark and pack to 3.5. Add once more to the 2 ml mark and pack down to 3. The columns are now packed with 1.5 ml celite/glycol mixture on top of 0.5 ml celite/water mixture. (See Appendix II for short column procedures).

Wetting the columns. Add 4 mls pure (nanograde) iso-octane to each column using a glass syringe. Attach hoses and allow nitrogen to flow through the columns. Stop when solvent level drops to within 1 mark of the celite. Add another 4 mls iso-octane to each column, but this time bring solvent just into celite. Nitrogen pressure may be as great as desired during this phase, but be careful not to dry out the celite. Should this occur, it must be rewet with enough iso-octane to wet it thoroughly. Columns that dry out badly will never run as rapidly as those not dried out.

Extraction Of Samples. While the columns are getting wet, aspirate the dichloromethane and extracted steroids (lower phase) with a disposable Pasteur pipet. Use a new pipet for each sample, and wear gloves. Gently blow bubbles through the plasma layer to avoid getting plasma into the pipet. Remove as much dichloromethane as possible without getting any plasma. Place dichloromethane into a 13x100 mm test tube, one tube for each sample. Place these tubes into a rack which fits the nitrogen evaporator. Evaporate the dichloromethane using the evaporator with the tubes in a water bath at 40oC.

COLUMN CHROMATOGRAPHY Day 2-3   ^ Back to top

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

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

Add 4 mls pure iso-octane and blow down under regulated pressure as above. The eluate is P. However, if P will be collected, use 4 mls of 2% ethyl acetate in iso-octane instead of pure iso-octane.

Add 4.5 mls 10% ethyl acetate in iso-octane as above. The eluate is DHT.

Add 4.5 mls 20% ethyl acetate in iso-octane. The eluate is T.

Add 4.5 mls 40% ethyl acetate in iso-octane. The eluate is E2. This step is optional and done only when assaying for E2.

Add 4.0 mls 50% ethyl acetate in iso-octane (4.5 mls if you skipped the 40% fraction). The eluate is B.

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

RADIOIMMUNOASSAY Day 4   ^ Back to top

Set up the appropriate number of small (10x75 mm), disposable test tubes in a rack. First arrange 12 pairs for the standard curve, with a space between the 3rd and 4th pair. Follow with a pair of tubes for each sample. If you have more than one spin of the centrifuge it is best to have each spin set up individually in racks.

For all steroids except corticosterone:

Setting up the samples. Vortex each tube before partitioning. Using an EP, pipet 200 µl of each sample into duplicate assay tubes. Either simultaneously or following, pipet 100 µl into a scintillation vial for the recovery. Scintillant will then be added to these vials. Cap and vortex.

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

Add 200 µl buffer to the B1 and B2 tubes, 100 µl buffer to remaining tubes except for S1, using the ER (5 ml tip set on 1). Add 100 µl cold steroid to each of the S1 and S2 pairs, using the HS. This will be 1000 pg for P, 500 pg for DHT, T, and E2, and 2000 pg for B. Vortex the S2 tubes, remove 100 µl and add to the S3 tubes. By continuing this serial dilution the concentration will be halved each time. Discard the last 100µl removed from the S9's. Go back and add 100µl buffer to B1-B3 and S1-S9 tubes (to bring up to same volume as samples). Gently mix (do not shake) hot steroid and antiserum prior to use. Using the ER, add 100 µl hot steroid to all tubes (standard curve plus unknown samples). Add 100 µl antiserum starting with the B3 tubes to all tubes (no antiserum in B1 and B2). Mix on the rack shaker and refrigerate overnight. Curves may be left in the refrigerator longer before adding charcoal, but overnight is the minimum time.

The standard curve contains:

• B1 buffer, labelled steroid = total cpm
• B2 buffer, labelled steroid = non-specific binding, background
• B3 buffer, labelled steroid, antiserum = maximum binding (Bmax)
• S1 100µl cold steroid, buffer added later, hot steroid,
• antiserum
• S2-9 buffer, serially diluted cold steroid, additional buffer, hot steroid, antiserum
• All unknowns 200 µl sample, hot steroid, antiserum

Note: When more than 48 samples occur, extra spins of the centrifuge are required. To correct for variations between spins, a new B2 and B3 are set up for each spin of the centrifuge.

Setting up corticosterone samples and curve:

The procedure for B is the same except different amounts are used.

Setting up the samples: Pipet 100 µl unknown sample into pairs of test tubes for radioimmunoassay. Pipet 200 µl unknown sample into a 7 ml scintillation vial to measure the recovery. This is the reverse of the procedure for all other steroids.

Setting up the standard curve: The procedure is identical except do not add the last 100 µl buffer to the B and S tubes following the serial dilution. This is because the unknowns only have 100 µl, so the volumes in the curve and the samples are identical. Otherwise, all instructions above apply

SEPARATION OF BOUND AND FREE COUNTS Day 5   ^ Back to top

Dextran-coated charcoal will be added to all tubes (except B1) and allowed to adsorb all unbound steroid. Tubes will be centrifuged, and the supernatant will be decanted into 7 ml scintillation vials for counting cpm.

• Run centrifuge through one complete cycle to cool it to 4oC.
  
• Add 0.5 mls dH2O or PBSG to B1's, using ER set on 2 with 12.5 ml tip. This can be done when the standard curve was set up on day 4.
  
• Mix charcoal very well by shaking and pour into a beaker with magnetic stir bar, place on mixer, and mix while adding 0.5 mls to remaining samples. Do no more than centrifuge will hold, in our case 120 tubes.
  
• Mix the racks gently and let stand for 12 minutes. Measure from time charcoal is added to last tube. This is a critical step. Set a timer and do not get distracted. Load centrifuge while waiting. Balance tubes both between and within buckets if there is less than a full spin.
  
• At 12 minutes centrifuge at 2000 rpm for 10 minutes.
  Decant supernatant into 7 ml scintillation vials, discarding tubes with charcoal pellet into LSA (radioactive waste) box.
  
• Add 4.5 mls scintillation fluid to all vials, cap, and vortex.
  
• Place on scintillation counter. This can be done immediately if a single-phase scintillant is used.
  
• To calculate plasma concentration of steroids see Appendix III.

CLEAN-UP   ^ Back to top

Glassware should be cleaned daily throughout the assay. Items are rinsed with tap H2O, soaked in a solution of Count-Off (from NEN) overnight, rinsed 3 times in tap H2O, then either submersed or rinsed 2 times in dH2O. Columns must be soaked before the celite can be removed (back pressure from a faucet attachment or a pipet washer) and then soaked in Count-Off. After final rinse, glassware is placed in a drying oven set below 40oC. If the temperature goes higher, absorption sites are set up on the glassware, causing assay problems. Should this occur, re-wash and dry.

Scintillation vials containing radioactive waste must be properly discarded. Follow guidelines established by your institution. For tritium waste, we empty the vials into gallon jugs, discard the caps after rinsing, wash the vials in Count-Off, and then dry and box for reuse or discard with regular glass garbage. The gallon jugs are cheaper to dispose of than the full vials. We do this even though we use a biodegradable scintillant (Ultima Gold - Packard Instrument Co.). Regulations regarding putting biodegradable scintillants down the sink vary from one institution to another. You must check with your own institution about proper disposal.
Each person is responsible for disposing of his/her vials in a timely manner. This should be immediately following a successful run on the scintillation counter and reading results. If all vials have counted then they can be discarded.

APPENDIX I - DIRECT ASSAYS   ^ Back to top

When separation of steroids in a plasma sample is unnecessary, a direct assay may be performed without columns. Generally this is only done with corticosterone (B), estradiol (E2), and progesterone (P). Other steroids have cross-reactions with the antisera and must first be separated from each other.

To perform a direct assay for corticosterone, E2 or T/DHT:

• Set up samples and recoveries as usual.
• Add dichloromethane, mix, allow to stand for 2 hours or more,
• aspirate the dichloromethane, and evaporate.
• Add buffer and leave overnight or shake for 45 min.
• Set up curves and recoveries as usual; remaining procedures are identical. Only the column chromatography has been omitted.

To perform a direct assay for E2 or P:M

• Set up samples and recoveries as usual.
• Add 4 mls ethyl ether, mix, allow to stand for 10 minutes, and then follow the ether extraction procedure for progesterone.
• Following ether extraction, evaporate, add buffer and proceed as above.

"Little B Assays"   ^ Back to top

When assaying for B and you have very small plasma volumes, you can do a "little B assay." The procedure is the same as for corticosterone above except:

• Use 5-25µl plasma, bring total volume to 200 µl with dH2O.
• Add 4 mls dichloromethane.
• Perform usual extraction and evaporation.
• Add 550µl buffer to sample tubes.
• Pipet samples and recoveries as for T, etc. - e.g. 200 µl to paired sample tubes, 100 µl to recoveries, and proceed as usual.
• Set up standard curve as for T, etc. After serial dilutions have been performed go back and add 100 µl buffer to all B and S tubes.
• Final calculations will be as for T, etc.

APPENDIX II - SHORT COLUMN CHROMATOGRAPHY   ^ Back to top

Short columns may be used if not assaying for both B and E2. The differences from a regular assay are:

• Pack the water trap to < 1 mark below the 4.5 mark on the column.
• Pack the celite/glycol mix to the 4 ml mark.
• Wet columns once with 4 mls pure iso-octane.
• Add samples to columns with 0.5 mls of 10% ethyl acetate in iso-octane.
• There is no rinse.

Fractions: For P, DHT, T, B.

When samples have been added to columns and blown down into celite with regulated nitrogen pressure:

• Add 2 mls pure iso-octane = P.
• Add 1.5 mls 10% ethyl acetate in iso-octane = DHT.
• Add 2.0 mls 20% ethyl acetate in iso-octane = T.
• Add 2.5 mls 50% ethyl acetate in iso-octane = B.

For P, DHT, T, and E2:

• As above except add 2.5 mls 40% ethyl acetate in iso-octane for E2 and skip the 50% fraction.
• Continue as usual for evaporation and setting up curves.

APPENDIX III - CALCULATIONS

Unless you have access to a scintillation counter that calculates and draws the standard curve and then calculates doses, you must generate the curve and then estimate doses from the curve for your unknown samples.

To generate the curve:
Average the values from the pairs of B2's-S9's. Subtract the B2 value from the B3-S9. Divide each new S value by the new B3 value. This is the % bound.

% bound = Sx-B2
B3-B2

For each S value, draw the % bound on semi-log paper and draw the curve.
Average the values from the pairs of your unknown samples and calculate the % bound exactly as you did for the S1-S9. Plug this value into the curve and estimate pg of steroid in your sample.

To calculate final plasma concentration of steroid

For P, DHT, T, E2, and "little B":

value from curve (pg) x 2.75 1000
Plasma concentration = ______________________ X ________
(pg/ml) % recovery (decimal) * µl plasma

Example:
Estimated dose from curve = 30 pg
Recovery = 0.764
Plasma volume = 150µl

30 x 2.75 1000
Plasma concentration = _______ X ____ = 719.9 pg/ml
.764 150
For B (done in the usual manner, sometimes called "big B')

value from curve (pg) x 10 1000
plasma concentration = _____________________ X ________
(pg/ml) % recovery (decimal) µl plasma

Calculation of recoveries:

For P, DHT, T, E2, little B (when recovery = 100µl):
Divide total cpm (taken from vial with 20µl of hot steroid) by 5.5. Divide all cpm from recoveries by this value.

For B (big B):
Divide total cpm by 5. Divide all cpm from recoveries by this value.

APPENDIX IV - REAGENTS   ^ Back to top

Steroid Assay Buffer - PBSG
(0.1M, pH 7.0)

NaH2PO4 : H2O 16.14 gm

Na2HPO4 : 7H2O 49.05 gm

NaCl 27.00 gm

gelatin 3.00 gm

NaN3 3.00 gm

dH2O 3 L

Mix in 4 L jug, and place on heated, magnetic stirrer. Heat over low heat while stirring until gelatin is dissolved. Refrigerate.

Dextran-coated Charcoal

Charcoal 2.5 gm
Dextran T-70 0.25 gm
PBSG 1 L

Place charcoal and dextran in bottle. Add PBSG and shake well. Refrigerate.

Scintillation Liquid

We use Ultima Gold, a biodegradable, single phase scintillant from Packard Instrument Co. (800-323-1891). It can handle a 20% aqueous load without forming precipitates.

APPENDIX V - PROGESTERONE EXTRACTION   ^ Back to top

To extract progesterone, you must use ethyl ether (we use Mallinckrodt brand). Add 4 mls ethyl ether to each centrifuge tube in the hood. Vortex at a low speed. Let sit for 10 minutes. Have a Dewar's thermos with dry ice and methanol or acetone ready. Dip the tip of each centrifuge tube into this cold liquid for about 15 seconds, freezing the plasma. Decant the ether extract into 13x100 mm test tubes and place in a rack for the evaporator. When all tubes have been done, place the rack in the H2O bath at room temperature and evaporate under nitrogen.

If cortisol is not being determined with progesterone, the ether extraction will be sufficient. Ethyl ether extracts all steroids except cortisol.

When running fractions of ethyl acetate in iso-octane on the columns, after the samples have been added to the columns and blown down, add 4 mls of 2% ethyl acetate in iso-octane instead of 4 mls of pure iso-octane. This brings most of the progesterone off in this fraction. Otherwise it tends to come off in both the 0% and the 10% fractions.

RIA SHORT SHEET   ^ Back to top

Sample preparation: Measure plasma samples into centrifuge tubes. Set up blanks and standard tube (50 µl cold steroid for each steroid being assayed).
Add 20 µl hot steroid to all tubes except blanks and a scintillation vial (for each steroid being assayed).
Bring all volumes to 400 µl with dH2O.
Let equilibrate overnight in refrigerator.
Add 5 ml dichloromethane and vortex.
Let sit > 2 hours.

Columns: Heat celite > 24 hours at 1000°F. Cool and weigh.
Water trap (6 gm:2 ml). Pack to 4.5 mark.
Glycol phase (6 gm:3 ml of 1:1 glycol mix). Pack to 3 mark in 0.5 ml increments.
Wet columns with iso-octane.

Extraction: Aspirate dichloromethane (lower phase) with disposable pipet into 13x100 mm test tubes in rack for evaporator. Evaporate under nitrogen in 40°C water bath.

Add samples to columns. Add 0.5 mls 10% ethyl acetate in iso-octane to tubes, whirl mix, pipet onto columns with disposable pipet. Rinse with another 0.5 mls and add to column. Run liquid into celite with regulated nitrogen pressure no faster than 1 drip/6 seconds.

Partition chromatography: Add appropriate fractions of ethyl acetate in iso-octane under regulated nitrogen pressure and collect or discard eluate:

0% 4.0 mls P (2% if collecting P)
10% 4.5 mls DHT
20% 4.5 mls T
40% 4.5 mls E2
50% 4.0 mls B

Evaporate liquid and add buffer: Evaporate under nitrogen in 40°C H2O bath.
Add 550µl buffer to all but B, 1000 µl to B. Let sit overnight at 4°C.

Partitioning samples: For all except B: place 200µl in paired sample tubes, 100µl in scintillation vials for recoveries. For B place 100µl in paired sample tubes, 200µl in recoveries.

Standard curve: Refer to procedure. Refrigerate overnight.

Separation and counting: Add 0.5 mls charcoal (H2O to B1). Let sit 12 minutes. Load centrifuge while waiting.
Centrifuge for 10 minutes at 2000 rpm.
Decant into scintillation vials.
Add scintillant, cap, mix.
Count on scintillation counter.