1. Radioimmunoassay
Definition: A Binding Assay ...
in which the binder is an antibody (*)
hi h radioactivity (#) t thwhich uses radioactivity (#) to measure the
amount of bound and/or free antigen
d l l b l d ll d " "Radioactively labeled antigen is called "tracer"
Radioactive isotopes are usually 3H (beta) or
125I (gamma)
(*) Other examples of binder molecules include ...
#(#) Alternative labels are ...

2. Radioimmunoassay: pros and cons
PRO: versatility : using the same principle, almost
b l l b dany biomolecule can be assayed
fast (usually 2 days or less)
sensitive (comparable to the most sensitive
bioassays, that is < ng/ml)y g
large capacity : thousands of samples/day
specific (antibody-dependent)specific (antibody dependent)
CON: use of radioactivity: hazardousCON: use of radioactivity: hazardous
expensive equipment (gamma or beta
t )counter)

3. The principle of RIA
The amount of Ab per tube is kept constant, the amount of
antigen added (known or unknown) is the variable parameter.
The added antigen will be distributed between a bound (B) andThe added antigen will be distributed between a bound (B) and
a free (F) fraction. This distribution is governed by the
association constant (KA)of the Ab:
Ab + Ag AgAb and K = [AbAg] /[Ab][Ag]
Ab
B F
Ag Say : K=1
And: [AbAg]=[Ab] =[Ag]= 1 (total Ag input = 2)
Result: B/F = 1
Say : total Ag input = 4Ag
b
S y : g p
Then : [AbAg] AND [Ag] will increase
E.g.: [AbAg] = 1.2 and [Ag] = 2.8 ( [Ab] = 0.8 )
Result : B/F = 0 43
B F
Ag
Ab
Result : B/F = 0.43

4. The principle of RIA (cont.)
Conclusion: If total Ab input is kept constant, the
value of B/F is a measure for the total Ag input
To measure this distribution B-F , a small but constant amount
g p
,
of labeled antigen ("tracer") is added to the reaction.
Eventually, there will be a competition reaction between thisy p
small but constant amount of "tracer" and the "cold" antigen
for a limited amount of antibody.
Ab Ag* Ab F Ag* Ab
B
F Ag*
B F
g
B
g
Ag
B
Agg

5. Measurement of Bound/Free Tracer
Ab
B
F Ag*
Ag
Since B + F = Ag* = constant, measurement ofg ,
either B OR F is sufficient.
Usually, B is measured by capture of the Ag-Ab complex.
This can be achieved by a solid-phase secondary Ab
or by lattice formation in solution.
super-
decant2nd ab
natant
precipitate
F
B precipitate
B count

6. Step 1 : The Antibody-Dilution Curve
Purpose: To determine the optimal amount of antibody to be
used typically enough to bind approx 50 % of the addedused, typically enough to bind approx. 50 % of the added
tracer (which is the same in each tube).
100
% B
5050
0
x
log [Ab]0
E.g. :
1/1,000,000 1/50,000 1/1,000polyclonal
serum

7. Step 2 : The Standard Curve
Purpose: To construct a binding inhibition curve based on
k ( d d) f i f i i l iknown (standard) amounts of antigen, for use in interpolation
of unknown samples.
B 0 = approx. 50 % of total added tracer (T)
% B 0
100
f l
Y
useful assay
range
0
x
0 2 4 8 16 32 64 128
log [Ag] (ng/ml)
0 2 4 8 16 32 64 128

8. Requirements for the development of an RIAq p
1. Pure antigen : for - standards (μg),
- tracer production (tens of μg)
- Ab production (hundreds of μg)
2. Tracer : self-made or commercial.2. Tracer : self made or commercial.
3. Specific, high-affinity Antibody : self-made or
commercial.
4 A method to separate bound and free antigen4. A method to separate bound and free antigen.
5. (Optional) : A system to extract the antigen from the
sample.

9. Antibody choiceAntibody choice
Above all, antibodies with high intrinsic affinity are needed.
RIA i li id h t h i ti bi di tRIA is a liquid phase technique: cooperative binding can not
make up for poor affinity.
O l th f ll t f tib d i ld itiOnly the use of small amounts of antibody yields a sensitive assay,
because a large amount of antibody would require a large amount
f ti t ti bl hift th ilib i (B/F)of antigen to noticeably shift the equilibrium (B/F).
AND
O l hi h ffi it tib di bl t bi d 50 % f th tOnly high affinity antibodies are able to bind 50 % of the tracer
when used at low concentrations.
P l l l i ld ll b tt i l t th b tPolyclonals yield usually better signal strength, but may con-
tain Abs of unwanted specificity.
Pooled monoclonals have both good specificity and signal strengthPooled monoclonals have both good specificity and signal strength.

10. RIA Tracers
1. Internally labeled molecules
T i ll t iti ( H) i th l b l ti CTypically, tritium (3H) is the label, sometimes 14C.
Usually purchased commercially.
Used only for small molecules like steroids or drugs.
Pro : the tracer immunologically behaves exactly asPro : the tracer immunologically behaves exactly as
the cold hormone, thus theoretically perfect.
Con : beta radiation is weak and therefore more difCon : beta-radiation is weak and therefore more dif-
ficult to measure, thus practically cumbersome.
B h l i hBeta rays have low penetrating power: the
radioactive sample needs to be mixed with a scintillator
fluid; the produced light is measured by use of a photofluid; the produced light is measured by use of a photo-
multiplier ("beta-counter").

11. RIA Tracers (cont.)
2. Externally labeled molecules
Typically, 125I is the label.Typically, 125I is the label.
Pro : often produced in the research lab itself.
Pro : gamma radiation has high penetrating powerPro : gamma-radiation has high penetrating power,
is therefore easy to measure, thus practical to use.
Con the tracer immunologically does not alwaysCon : the tracer immunologically does not always
behave exactly as the cold hormone, due to iodination
damagedamage.
Con : shelf-life of iodinated protein is < 4 weeks.
U ll I ill t k th l f h d tUsually, 125I will take the place of a hydrogen atom on
on the ring of tyrosine.
S ti di ti l l b l d l l d t bSometimes, a radioactively labeled molecule needs to be
conjugated to the protein.

12. The virtues of a good radioimmunoassay
PRECISION : ≈ reproducibility; characterized by
low inter-assay and intra-assay variability.y y y
(Both values need to be < 10%)
ACCURACY are the figures approaching the realACCURACY : are the figures approaching the real
concentration? (Use an independent approach to
verify e g a physicochemical technique)verify e.g. a physicochemical technique)
SENSITIVITY : how little can still be detected?
(Can be enhanced by pre-incubating the cold
hormone with the Ab, prior to tracer addition)
SPECIFICITY : lack of cross-reaction with related
molecules. Dilution curves of samples and standardsp
need to be parallel!

13. RIA specificity (non-specificity)
% B 0
A B
Parallel curves : cross-reactivity can be calculated :
(10 %)
100
% B 0
50
A B
standard curve
dilution curve of
cross-reacting substance
0
x
l [A ] ( / l)
0 2 4 8 16 32 64 128
20 200 log [Ag] (ng/ml)20 200
Non-Parallel curves : quantitation impossible!q p
100
% B 0
A B dilution curve of
cross-reacting substance
50
g
standard curve
0
log [Ag] (ng/ml)
0 2 4 8 16 32 64 128