Week 9 radioimmunoassay

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