week 9 radioimmunoassay
Post on 11-Nov-2014
969 Views
Preview:
DESCRIPTION
TRANSCRIPT
Radioimmunoassay
Definition: A Binding Assay ...
in which the binder is an antibody (*)hi h radioactivity (#) t th which uses radioactivity (#) to measure the
amount of bound and/or free antigend 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 ...
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 sensitivebioassays, that is < ng/ml)y glarge capacity : thousands of samples/dayspecific (antibody-dependent)specific (antibody dependent)
CON: use of radioactivity: hazardousCON: use of radioactivity: hazardousexpensive equipment (gamma or beta
t )counter)
The principle of RIA
The amount of Ab per tube is kept constant, the amount ofantigen added (known or unknown) is the variable parameter.The added antigen will be distributed between a bound (B) and 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 AgAb and K = [AbAg] /[Ab][Ag]
AbB F
Ag Say : K=1And: [AbAg]=[Ab] =[Ag]= 1 (total Ag input = 2)Result: B/F = 1
Say : total Ag input = 4AgbS y : g pThen : [AbAg] AND [Ag] will increaseE.g.: [AbAg] = 1.2 and [Ag] = 2.8 ( [Ab] = 0.8 )Result : B/F = 0 43
B F
AgAb
Result : B/F = 0.43
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 psmall but constant amount of "tracer" and the "cold" antigen for a limited amount of antibody.
Ab Ag* Ab F Ag* AbB
F Ag*
B Fg
Bg
Ag
B
Agg
Measurement of Bound/Free Tracer
AbB
F Ag*
Ag
Since B + F = Ag* = constant, measurement of g ,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 Abor by lattice formation in solution.
super-decant2nd ab
natant
precipitate
F
B precipitateB count
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 added used, typically enough to bind approx. 50 % of the added tracer (which is the same in each tube).
100% B
5050
0 xlog [Ab]0E.g. :
1/1,000,000 1/50,000 1/1,000polyclonal serum
Step 2 : The Standard Curve
Purpose: To construct a binding inhibition curve based on k ( d d) f i f i i l i known (standard) amounts of antigen, for use in interpolation of unknown samples.
B 0 = approx. 50 % of total added tracer (T)
% B 0100
f l Y
useful assay range
0 x0 2 4 8 16 32 64 128
log [Ag] (ng/ml)0 2 4 8 16 32 64 128
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.
Antibody choiceAntibody choiceAbove all, antibodies with high intrinsic affinity are needed. RIA i li id h t h i ti bi di t RIA 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 iti Only 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 tracerwhen used at low concentrations.
P l l l i ld ll b tt i l t th b t Polyclonals yield usually better signal strength, but may con-tain Abs of unwanted specificity.
Pooled monoclonals have both good specificity and signal strength Pooled monoclonals have both good specificity and signal strength.
RIA Tracers1. 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 asthe 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 h Beta 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 photo fluid; the produced light is measured by use of a photo- multiplier ("beta-counter").
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 always Con : the tracer immunologically does not always behave exactly as the cold hormone, due to iodinationdamagedamage.Con : shelf-life of iodinated protein is < 4 weeks. U ll I ill t k th l f h d t Usually, 125I will take the place of a hydrogen atom onon the ring of tyrosine. S ti di ti l l b l d l l d t b Sometimes, a radioactively labeled molecule needs to be conjugated to the protein.
The virtues of a good radioimmunoassayPRECISION : ≈ reproducibility; characterized bylow 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 realconcentration? (Use an independent approach toverify 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 coldhormone with the Ab, prior to tracer addition)
SPECIFICITY : lack of cross-reaction with relatedmolecules. Dilution curves of samples and standardspneed to be parallel!
RIA specificity (non-specificity)
% B 0 A B
Parallel curves : cross-reactivity can be calculated :(10 %)
100% B 0
50
A Bstandard 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
gstandard curve
0
log [Ag] (ng/ml)0 2 4 8 16 32 64 128
top related