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1. 1. RadioimmunoassayDefinition: A Binding Assay ...in which the binder is an antibody (*)hi h radioactivity (#) t thwhich uses radioactivity (#) to measure theamount 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) or125I (gamma)(*) Other examples of binder molecules include ...#(#) Alternative labels are ...
2. 2. Radioimmunoassay: pros and consPRO: versatility : using the same principle, almostb l l b dany biomolecule can be assayedfast (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 betat )counter)
3. 3. The principle of RIAThe 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) andThe added antigen will be distributed between a bound (B) anda free (F) fraction. This distribution is governed by theassociation constant (KA)of the Ab:Ab + Ag AgAb and K = [AbAg] /[Ab][Ag]AbB FAg Say : K=1And: [AbAg]=[Ab] =[Ag]= 1 (total Ag input = 2)Result: B/F = 1Say : 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 43B FAgAbResult : B/F = 0.43
4. 4. The principle of RIA (cont.)Conclusion: If total Ab input is kept constant, thevalue of B/F is a measure for the total Ag inputTo measure this distribution B-F , a small but constant amountg 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" antigenfor a limited amount of antibody.Ab Ag* Ab F Ag* AbBF Ag*B FgBgAgBAgg
5. 5. Measurement of Bound/Free TracerAbBF Ag*AgSince 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 Abor by lattice formation in solution.super-decant2nd abnatantprecipitateFB precipitateB count
6. 6. Step 1 : The Antibody-Dilution CurvePurpose: To determine the optimal amount of antibody to beused typically enough to bind approx 50 % of the addedused, typically enough to bind approx. 50 % of the addedtracer (which is the same in each tube).100% B50500xlog [Ab]0E.g. :1/1,000,000 1/50,000 1/1,000polyclonalserum
7. 7. Step 2 : The Standard CurvePurpose: To construct a binding inhibition curve based onk ( d d) f i f i i l iknown (standard) amounts of antigen, for use in interpolationof unknown samples.B 0 = approx. 50 % of total added tracer (T)% B 0100f lYuseful assayrange0x0 2 4 8 16 32 64 128log [Ag] (ng/ml)0 2 4 8 16 32 64 128
8. 8. Requirements for the development of an RIAq p1. 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 orcommercial.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 thesample.
9. 9. Antibody choiceAntibody choiceAbove 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 notmake 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 amountf ti t ti bl hift th ilib i (B/F)of antigen to noticeably shift the equilibrium (B/F).ANDO 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 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. 10. RIA Tracers1. Internally labeled moleculesT 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 hBeta rays have low penetrating power: theradioactive sample needs to be mixed with a scintillatorfluid; the produced light is measured by use of a photofluid; the produced light is measured by use of a photo-multiplier ("beta-counter").
11. 11. RIA Tracers (cont.)2. Externally labeled moleculesTypically, 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 alwaysbehave 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 tUsually, 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 bSometimes, a radioactively labeled molecule needs to beconjugated to the protein.
12. 12. 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!
13. 13. RIA specificity (non-specificity)% B 0A BParallel curves : cross-reactivity can be calculated :(10 %)100% B 050A Bstandard curvedilution curve ofcross-reacting substance0xl [A ] ( / l)0 2 4 8 16 32 64 12820 200 log [Ag] (ng/ml)20 200Non-Parallel curves : quantitation impossible!q p100% B 0A B dilution curve ofcross-reacting substance50gstandard curve0log [Ag] (ng/ml)0 2 4 8 16 32 64 128