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Filename:ACTHRIA.doc Written by: J. Ciccotosto (033198) Edited by: D. Mains (010400)

Protocol:ACTH Radioimmunoassay

Include the following sets of tubes in each assay

Total Tubes (n=4).Take 100 l of hot mix and put into the longer plastic (12x75 mm) tubes, cap, and save to count next day.

Minus Ab (n=3)Take 3 by 100 l aliquots of hot mix BEFORE adding the antibody.

Minus Cold (n=3)Take 3 by 100 l aliquots of hot mix and process as per sample tubes.

Std Curve (n=10)Ranges from 20 pg to 10,000 pg.

Sample tubesDetermine the amount of sample per assay (between 100 to 200 l) and then bring volume of sample to 200 l with RIA buffer. Do up to 5 sets of serial 1:2 dilutions.

Hot Mix

Reagent / stock / final / dilution
125I-ACTH(1-39) / 105 cpm/tube / 104 cpm/tube / 0.1 l per tube
Rabbit IgG / Neat / 2 l/tube / 1/50

Before adding Ab, take 3 by 100 l aliquot samples for Minus Ab tubes.

Ab Kathy / 1:10 / 1:15,000 / 1/1,500
  1. Add the hot mix to the remaining tubes.
  1. Incubate all other samples overnight at 4 0C for at least 12 hrs. (Ab Kathy benefits from going 24-30 hours).
  1. In the morning, add GAR (50l, diluted 4 l of Biogenesis stock plus 46 l of RIA buffer per tube) to all tubes and incubate at 4 0C for another 2 hrs.
  1. About 40 min before the end of the 2 hours with GAR: Turn on the centrifuge and Lock the door and spin for 30 min so that it cools to 4 0C.
  1. After the 2 hr incubation (should see cloudiness in tubes), add 1 ml of RIA buffer to all tubes.
  1. Put tubes into blue racks and spin at top speed (about 2700 rpm) for 30 min.
  1. Using a pasteur pipette, aspirate supernatant from each tube (careful not to aspirate the pellet. If pellet is aspirated, put back into tube and spin tube again).
  1. Place the plastic tubes into glass tubes. It is easiest for calculations if you count tubes in the order Totals : -Ab : -Cold : Standard : Samples
  2. Count in gamma counter using the RIA (2 min) program. Transfer the data set to a floppy disk and import into Excel on the calculations computer.
  1. Calculate the assay using the Excel RIA (Packard) program.

125I-ACTH(1-39)

10Ci stock (Amersham). There are 2 types, Tyr2 or Tyr23 iodinations.

10 Ci = 10 Ci x 2x106 cpm/Ci = 20x106 cpm

Dissolve radioactive sample in 200 l of (1% Acetic Acid/0.1% Triton X-100)

Final concentration is 105 cpm/tube. There need about 0.1 l trace ACTH per assay tube.

ACTH(1-39) standard curve

Stock concentration is 10 g/ml. Dissolved in 0.1 % Acetic Acid, 0.1% Triton X-100.

Standard Curve in each assay ranges from 20 pg to 10,000 pg.

To set up the standard curve, label tubes 1 to 10 (see table below).

To tube #1, add 198 l of RIA buffer. To tubes #2 to 9 add 100 l RIA buffer.

To tube #1, add 2 l stock standard (10 g/ml) and mix well.

Then take 100 l from tube #1 and add to tube #2.

Repeat serial dilution to tube #10 and then discard 100 l after the last tube, #10.

Tube # / ACTH (pg)
1 / 10,000
2 / 5,000
3 / 2,500
4 / 1,250
5 / 625
6 / 313
7 / 156
8 / 78
9 / 39
10 / 20

Antibody

KathyDirected against the CT of ACTH. This antibody sees ABI, ACTH, g-ACTH, and CLIP but NOT intact POMC. A stock of 1:10 is kept in the refrigerator. Assay final concentration is often 1:15,000 (higher sensitivity with 1:25,000 and longer incubation times).

RIA Buffer (1L)

50 mM NaPi, pH 7.6, 0.1% Triton X-100

NaH2PO4...... 6.9 g

Triton X-100...... 1ml

Water...... 999 ml

Adjust to pH 7.6 with 10N NaOH

Chemical / Vendor / Catalog #
125I-Tyr2-ACTH(1-39), 10 Ci / Amersham / IM.216
125I-Tyr23-ACTH(1-39), 10 Ci / Amersham / IM.183
Triton X-100 / Biorad / 161-0407
Sodium Phosphate, Mono (NaH2PO4) / J.T. Baker / 3818-05
Goat Anti Rabbit / Biogenesis
hACTH(1-39) / CIBA

ACTH RIA ASSAY COVER SHEET

Assay Date:______Assay Number:______

Experiment:______

______

______

______

______

HOT MIX

Total Number of samples = ______

A. Total Hot Mix Volume 100 l x ______samples= ______l

Label [125I-ACTH(1-39)] (105 cpm/l Stock; 103 cpm/tube final)
B. Label activity date:______0.1 x______samples=______µl

C. Rabbit IgG (2l/tube)2 x______samples=______µl

Volume (B+C)=______µl

D. RIA BufferVolume A-(B+C)=______l

Before adding Ab, take 3 by 100 l aliquot samples for Minus Ab tubes.(-300 l)

Final Vol=______l

Ab_KathyStock (1/10) final (1/15K) 1/1500 x samples=______l

Add 100 l to each tube. Incubate all other samples overnight at 4 0C for at least 12 hrs.

Ab reaction time Start: ______Stop: ______Total Time: ______

GAR (3l/tube)4 x samples=______µl

RIA buffer46 x samples=______µl

Add 50 l of diluted GAR to each tube. Incubate at 4 0C for at least 2 hrs.

GAR reaction time Start: ______Stop: ______Total Time:______

ACTH RIA procedures

  1. Count the 4 Total Tubes on the gamma counter.
  1. Incubate all other samples overnight at 4 0C for at least 12 hrs.
  1. In the morning, add GAR (50l) to all tubes and incubate at 4 0C for another 2 hrs.
  1. Turn on the centrifuge (Lock the door) and spin for 30 min so that it cools to 4 0C.
  1. After the 2 hr incubation (should see small white pellet), add 1 ml of RIA buffer to all tubes.
  1. Put tubes into the blue centrifuge racks and spin at top speed (about 2700 rpm) for 30 min.
  1. Using a pasteur pipette, aspirate supernatant from each tube
    (careful not to aspirate the pellet. If pellet is aspirated, put back into tube and spin tube again).
  1. Place the plastic tubes into glass tubes and get gamma counter ready for counting samples.
  1. Before counting tubes, change the longer barrels on the gamma counter with the shorter barrels.
  1. Calculate the assay using the Excel RIA program.

Eipper/Mains Protocol Manual