6.4.02 SBF
ImmunoChem Double Antibody: CORTICOSTERONE 125I RIA KIT
(protocol adapted for Vazquez lab from ICN)
I.
Principle of Test
In this assay, a limited amount of specific antibody (Ab) is reacted with the corresponding
hormone (*CORT) labeled with a radioisotope. Upon addition of an increasing amount of
endogenous hormone (CORT), a correspondingly decreasing fraction of *CORT is bound to the
antibody. The precipitant solution (mixture of PEG and Goat anti-rabbit gamma globulins in
TRIS buffer) will immediately precipitate all of the antibody bound antigen. After separation of
the bound from the free *CORT (by centrifugation) the amount of radioactivity in the precipitant
is evaluated and used to construct a standard curve against which the unknown samples are
measured. (Thus, the higher the number of counts (CPM), the more *CORT is bound and the
lower the level of endogenous CORT).
II.
Equipment and Reagents Required
1) Reagents supplied by kit:
a) Steroid Diluent
b) Anti-Corticosterone
c) Corticosterone Calibrators (25, 50, 100, 250, 500 and 1000 ng/mL)
d) Precipitant Solution
e) Corticosterone-125I Derivative
2) Pipets and/or repeater
3) 12 x 75 mm polypropylene conical-bottomed tubes for RIA
4) Test tube racks
5) Vortex mixer
6) Centrifuge (capable of 2300-2500 rpm (1000 x g)
7) Absorbant paper for blotting or vacuum for aspiration
8) Gamma Counter
III.
Assay Procedure
A. Assay Preparations
1) Bring reagents to room temperature prior to use. (Thaw samples!)
2) Set up assay in consecutively numbered 12 x 75 mm tubes (all in duplicate)
3) Add solutions in the order indicated in the protocol. Pipet all reagents directly from
shipping vials.
B. Assay Steps
1) Dilute rat or mouse serum 1:200 with STEROID DILUENT by taking 10 ul of sample to
2.0 mL of diluent.
2) Add 0.3 mL (300 ul) STEROID DILUENT to tubes 1 and 2 (NSB tubes).
3) Add 0.1 mL (100 ul) STEROID DILUENT to tubes 3 and 4 (0 tubes).
4) Add 0.1 mL (100 ul) CORTICOSTERONE CALIBRATORS* (25 ng/mL-1000 ng/mL)
to tubes 5-16.
*Concentration of calibrators are expressed as serum equivalent and have already included the
dilution factor. Results can be read directly from calibration curve if the recommended dilution of
(1:200) is followed.
5) Add 0.1 mL (100 ul) DILUTED (1:200) CONTROLS and DILUTED (1:200)
RAT/MOUSE SERUM to tubes 17 to end of assay.
6) Add 0.2 mL (200 ul) CORTICOSTERONE-125I (blue reagent) to all tubes.
- NOTE: 125I TRACER MUST BE ADDED BEFORE ANTI-SERUM
7) Add 0.2 mL (200 ul) ANTI-CORTICOSTERONE (yellow reagent) to tubes 3 to end of
assay.
- NOTE: DO NOT ADD ANTISERUM TO NSB TUBES
8) Vortex mix ALL assay tubes and incubate at room temperature (22-25ºC) for 2 hours.
9) After incubation, add 0.5 mL (500 ul) PRECIPITANT SOLUTION (red reagent) to all
tubes.
10) Vortex THOROUGHLY.
11) Centrifuge all assay tubes at 23-2500 rpm (1000 g) for 15 min. (no need to refrigerate
centrifuge)
12) Aspirate the supernatant in the hot room.
13) Count the precipitate in the gamma counter.
IV.
Using the Gamma Counter
1) Turn on the gamma counter and the printer using the power switch on the right side of each
instrument.
2) If the counter has not been calibrated recently, you must do so using a 125I sample with
minimum CPM of 25,000. See Chapter 2 in the manual for details. Calibration AND
Normalization should be performed monthly.
3) Find and/or create a proper protocal to use on the gamma counter. Protocol #s 14 and 18
should work for I125 RIAs, but you will need to check and correct for the number of
standards used. You can do this using the "tube display" option.
4) The counter moves cassettes clockwise around the sample changer deck until a protocol clip
is read. So, place your first rack with the correct protocol clip on it on the left-hand side of
the counter. Place the rest of the cassettes in front of it so that it will read the samples in the
order intended - remember, it reads CLOCKWISE! Place the STOP clip on the last rack so
the counter will not continuously count and waste paper!!!
5) Once your samples/racks are placed in the correct order on the counter, from the directory
screen go to "SC Commands" (F2) and then to "Count" (F5). The machine should take over
from here and generate a pretty standard curve to read your samples off of.