Radioimmunoassay
Definition: A Binding Assay ...
in which the binder is an antibody (*)
which
hi h uses radioactivity (#) tto measure the
th
amount of bound and/or free antigen
Radioactively
d
l labeled
l b l d antigen is called
ll d ""tracer""
Radioactive isotopes are usually 3H (beta) or
125I (gamma)
(*) Other examples of binder molecules include ...
(#)
# Alternative labels are ...
Radioimmunoassay: pros and cons
PRO:
versatility : using the same principle, almost
any bbiomolecule
l l can bbe assayedd
fast (usually 2 days or less)
sensitive (comparable to the most sensitive
y that is < ng/ml)
g
bioassays,
large capacity : thousands of samples/day
specific (antibody
(antibody-dependent)
dependent)
CON:
use of radioactivity: hazardous
expensive equipment (gamma or beta
counter)
t )
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) and
a free (F) fraction. This distribution is governed by the
association constant (KA) of the Ab:
Ab + Ag z AgAb and K = [AbAg] /[Ab][Ag]
Ag
Ab
B F
Ag
Abb
B
F
Say : K=1
And: [AbAg]=[Ab] =[Ag]= 1 (total Ag input = 2)
Result: B/F = 1
S y : total Agg input
Say
p =4
Then : [AbAg] AND [Ag] will increase
E.g.: [AbAg] = 1.2 and [Ag] = 2.8 ( [Ab] = 0.8 )
Result : B/F = 0.43
0 43
The principle of RIA (cont.)
Conclusion: If total Ab input is kept constant, the
p
value of B/F is a measure for the total Agg input
To measure this distribution B-F , a small but constant amount
of labeled antigen ("tracer") is added to the reaction.
Eventually,
y there will be a competition
p
reaction between this
small but constant amount of "tracer" and the "cold" antigen
for a limited amount of antibody.
Ab
g
Ag*
B F
Ab
B
g
F Ag*
Agg
Ab
B
F
Ag*
Ag
Measurement of Bound/Free Tracer
Ab
B
F Ag*
Ag
Since B + F = Ag*
g = constant,, measurement of
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.
2nd ab
supernatant
F
B
precipitate
decant
count
Step 1 : The Antibody-Dilution Curve
Purpose: To determine the optimal amount of antibody to be
used typically enough to bind approx.
used,
approx 50 % of the added
tracer (which is the same in each tube).
%B
100
50
0
E.g. :
polyclonal
serum
0
1/1,000,000
x
1/50,000
log [Ab]
1/1,000
Step 2 : The Standard Curve
Purpose: To construct a binding inhibition curve based on
k
known
((standard)
d d) amounts off antigen,
i
for
f use iin interpolation
i
l i
of unknown samples.
B 0 = approx. 50 % of total added tracer (T)
%B0
100
useful
f l assay
range
Y
0
0
2
4
8
16
x
32
64
128
log [Ag] (ng/ml)
Requirements
q
for the development
p
of an RIA
1. Pure antigen : for - standards (μg),
- tracer production (tens of μg)
- Ab production (hundreds of μg)
2. Tracer : self-made
self made or commercial.
3. Specific, high-affinity Antibody : self-made or
commercial.
4 A method to separate bound and free antigen
4.
antigen.
5. (Optional) : A system to extract the antigen from the
sample.
Antibody choice
Above all, antibodies with high intrinsic affinity are needed.
RIA iis a liliquid
id phase
h ttechnique:
h i
cooperative
ti bi
binding
di can nott
make up for poor affinity.
Only
O
l th
the use off smallll amounts
t off antibody
tib d yields
i ld a sensitive
iti assay,
because a large amount of antibody would require a large amount
off antigen
ti to
t noticeably
ti bl shift
hift th
the equilibrium
ilib i (B/F)
(B/F).
AND
Only
O
l hi
highh affinity
ffi it antibodies
tib di are able
bl to
t bind
bi d 50 % off the
th tracer
t
when used at low concentrations.
PPolyclonals
l l l yield
i ld usually
ll bbetter
tt signal
i l strength,
t
th bbutt may contain Abs of unwanted specificity.
Pooled monoclonals have both good specificity and signal strength.
strength
RIA Tracers
1. Internally labeled molecules
T i ll tritium
Typically,
t iti
(3H) is
i the
th llabel,
b l sometimes
ti
C
14C.
Usually purchased commercially.
Used only for small molecules like steroids or drugs.
Pro : the tracer immunologically behaves exactly as
the cold hormone, thus theoretically perfect.
Con : beta
beta-radiation
radiation is weak and therefore more dif
difficult to measure, thus practically cumbersome.
Beta rays have
B
h low
l penetrating
i power: the
h
radioactive sample needs to be mixed with a scintillator
fluid; the produced light is measured by use of a photophoto
multiplier ("beta-counter").
RIA Tracers (cont.)
2. Externally labeled molecules
Typically, 125I is the label.
Pro : often produced in the research lab itself.
Pro : gamma-radiation
gamma radiation has high penetrating power,
power
is therefore easy to measure, thus practical to use.
Con : the tracer immunologically does not always
behave exactly as the cold hormone, due to iodination
damage.
damage
Con : shelf-life of iodinated protein is < 4 weeks.
U ll 125I will
Usually,
ill ttake
k th
the place
l off a hhydrogen
d
atom
t on
on the ring of tyrosine.
S
Sometimes,
ti
a radioactively
di ti l llabeled
b l d molecule
l l needs
d tto bbe
conjugated to the protein.
The virtues of a good radioimmunoassay
PRECISION : ≈ reproducibility; characterized by
low inter-assayy and intra-assayy variability.
y
(Both values need to be < 10%)
ACCURACY : are the figures approaching the real
concentration? (Use an independent approach to
verify e.g.
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
p and standards
molecules. Dilution curves of samples
need to be parallel!
RIA specificity (non-specificity)
Parallel curves : cross-reactivity can be calculated :
A
%B0
100
(10 %)
B
dilution curve of
cross-reacting substance
standard curve
50
0
0
2
4
8
16
x
20
32
64
128
200
l [A
log
[Ag]] ((ng/ml)
/ l)
Non-Parallel curves : qquantitation impossible!
p
%B
A
B
dilution curve of
0
100
cross-reactingg substance
standard curve
50
0
0
2
4
8
16
32
64
128
log [Ag] (ng/ml)