Week 9 radioimmunoassay
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Week 9 radioimmunoassay






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Week 9 radioimmunoassay Week 9 radioimmunoassay Presentation Transcript

  • 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 ...
  • 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)
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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.
  • 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.
  • 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").
  • 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.
  • 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 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