Radio Immuno as Says

8/11/2019 Radio Immuno as Says

1/10

RadioimmunoassaysArticle Outline Overview

What do I need to run this assay?

Defnitions

Types o assays

o Saturation assay

o Competition assay

Calculations

Tips and FAs

!eerences

Custom la"elin# services

Overview

!adioimmunoassays $!IAs% use anti"odies to detect and &uantitate the amount o anti#en $analyte% in a sample' Theseassays are typically very sensitive and specifc' It is possi"le to detect as low as a ew pico#rams o analyte in thee(perimental tu"e when usin# anti"odies o hi#h a)nity $*d + ,- ./. ,-.,,0%' The "asic principle o radioimmunoassayis competitive "indin#1 where a radioactive anti#en $2tracer2% competes with a non.radioactive anti#en or a f(ednum"er o anti"ody or receptor "indin# sites' When unla"eled anti#en rom standards or samples and a f(ed amounto tracer $la"eled anti#en% are allowed to react with a constant and limitin# amount o anti"ody1 decreasin# amountso tracer are "ound to the anti"ody as the amount o unla"eled anti#en is increased'In our ,34 I and 56 radioimmunoassay 7its1 separation o the anti"ody.anti#en comple(es rom ree anti#en is achieved"y precipitation o the anti"ody."ound tracer with either a secondary anti"ody solution directed a#ainst the #enus orspecies specifc immuno#lo"ulins o the primary anti"ody1 or "y use o polyethylene #lycol' 8oth precipitators#enerally re&uire the presence o carrier immuno#lo"ulin' Ater centriu#ation1 the supernatant containin# the un"oundanti#en is decanted1 and the pellet containin# the anti"ody.anti#en comple( is counted in a scintillationcounter' !esults o"tained or the standards are used to construct a standard $dose.response% curve rom which theun7nowns are calculated "y interpolation'

Figure 1. Principle of a competitive bindingradioimmunoassay. Radiolabeled antigen ("tracer") added to an antibody specic to the antigen leads to formation of an antigen-antibodycomple. !nlabeled antigen from a sample or standard solution can also bind antibody leading to unlabeled antigen-antibody comple. #n theradioimmunoassay the amount of radiolabeled antigen (tracer) is held constant. #ncreasing amounts of unlabeled antigen in the sample $illcompete $ith tracer for binding to the antibody leading to more unlabeled antigen-antibody comple.

Fi#ure 3 illustrates how a limited amount o anti"ody "indin# sites contained in a test tu"e or microplate well caneither "ind unla"eled li#and $in this e(ample1 6epatitis 8 Surace Anti#en% or radiola"eled li#and' As the amount ounla"eled li#and increases1 there is conse&uently less radiola"eled li#and "ound' The unla"eled li#and can come romeither a 9cali"ration standard: or the sample that you are tryin# to measure'
http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Overviewhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-WhatdoIneedtorunthisassayhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Definitionshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Typesofassayshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Saturationassayhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Competitionassayhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Calculationshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-TipsandFAQshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Referenceshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Customlabelingserviceshttp://www.perkinelmer.com/Catalog/Category/ID/RIA%20Kitshttp://www.perkinelmer.com/Catalog/Category/ID/RIA%20Kitshttp://www.perkinelmer.com/Catalog/Category/ID/RIA%20Kitshttp://www.perkinelmer.com/Catalog/Category/ID/RIA%20Kitshttp://www.perkinelmer.com/CMSResources/Images/44-148810ria_main.jpghttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-WhatdoIneedtorunthisassayhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Definitionshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Typesofassayshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Saturationassayhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Competitionassayhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Calculationshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-TipsandFAQshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Referenceshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Customlabelingserviceshttp://www.perkinelmer.com/Catalog/Category/ID/RIA%20Kitshttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#Radioimmunoassays-Overview


2/10

Figure %. a) &ample containing a high amount ofantigen. 'he unlabeled antigen competes for binding to the antibody in the tube or $ell. b) &ample containing no or lo$ amounts of antigen.

ntibody is bound by the radiolabeled antigen (tracer).

top

What do I need to run this assay?

Assay Buffer

A typical "u;er mi#ht "e 4- m0 phosphate p6 < with -'=> aCl and -'-4> sodium a@ide' To minimi@enonspecifc anti#en 9stic7in#: to the reaction tu"es1 -'5> "ovine serum al"umin may "e added' Other additivesmay include ,- m0 DTA andBor -'4> Tween.3-'

Radiolabeled ,34I- or 5H- tracer. Two terms must be understood when optimizing an RIA tracer concentration

Specifc Activity This is the amount o radioactivity that is present on the la"eled anti#en $li#and%' The units

are usually CiBmmol or CiB#' 8y 7nowin# this value1 the mass o diluted tracer per unit volume in the assay is7nown' The ideal mass concentration o tracer in an assay is set at sli#htly "elow the saturatin# concentration orthe antiserum dilution used in the assay' Ideally1 5-.E-> o the la"eled li#and $@ero standard "indin#% should "e"ound in the a"sence o unla"eled li#and at the appropriate tracer mass and antiserum dilution' This providesoptimum assay sensitivity' !adioli#ands with a hi#h specifc activity are well.suited or li#and "indin# assays'Specifc activity indicates how much radioactivity there is per molecule o li#and1 and is usually #iven in units oCuries per millimole o li#and' Several o our,34I.la"eled li#ands are o;ered at ma(imum specifc activity $33--CiBmmol i one ,34I la"elin# site is availa"le1 -- CiBmmol i two ,34I la"elin# sites are availa"le1 etc'%' This indicatesthat virtually every molecule o li#and provided in the stoc7 vial is radiola"eled' For tritiated li#ands $56 li#ands%1you should ideally pic7 a li#and that has a specifc activity a"ove 3- CiBmmol' The ma(imum theoretical specifcactivity per tritium is 3= CiBmmol $Curies per millimole o tritium%' Specifc activities a"ove this value indicate that onavera#e1 each molecule o li#and has at least one tritium'

Activity Concentration This is the concentration1 usually in CiBmG1 o radioactivity per unit volume o solvent

or diluent' It has little to do with the actual optimi@ation o tracer mass1 "ut serves as a re&uired measurement othe correct amount o counts added to the assay' This is important or the user to 7now "ecause the calculation onon.specifc "indin# $S8%1 @ero standard "indin# and all other "indin# measurements depend on this'

HIn most !IA 7its the tracer concentration has already "een optimi@ed1 and all that is necessary is to perorm therecommended dilution in assay "u;er and use the appropriate volume per assay tu"e'

HHipets andBor pipet tips used to transer the tracer solution must "e o polypropylene or siliconi@ed #lass' Do not

use those made o unsiliconi@ed #lass1 as there may "e stic7in# issues'

There are an additional ew actors to 7eep in mind when selectin# a tracer or your assay

on.specifc "indin# 6ydropho"ic tracers will #enerally show hi#her non.specifc "indin#' 8y

includin# 8SA1 certain salts or deter#ents in the assay "u;er you can help to reduce non.specifc "indin#' I thestoc7 radiochemical is pac7a#ed in a silani@ed vial $reer to the tech data sheet%1 this may indicate the li#and issomewhat hydropho"ic

6i#h purity Ideally1 the tracer should have a radiochemical purity a"ove =->' !adiochemical

purity decreases over time1 and the actual rate o this de#radation accelerates over time' !adiochemical purityand de#radation rates or our radiochemicals can "e ound on each lot.specifc technical data sheet'
http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#tophttp://www.perkinelmer.com/coahttp://www.perkinelmer.com/coahttp://www.perkinelmer.com/CMSResources/Images/44-148811ria_main_2.jpghttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#tophttp://www.perkinelmer.com/coahttp://www.perkinelmer.com/coa


3/10

6i#h selectivity The more selective the tracer is or your anti"ody or "inder1 the "etter your

data will "e' 6i#h selectivity indicates the tracer will mostly "e reco#ni@ed "y appropriate anti"ody "indin#sites''

Sta"ility I you will need to use your radiola"eled tracer over an e(tended period o time1

sta"ility may "e a actor or you' ,34I.la"eled li#ands should #enerally "e used within one to two months othe manuacture date' Tritiated li#ands should usually "e used within 5.E months o manuacture dateJhowever1 there are e(ceptions to this' De#radation rates and manuacturin# dates can "e ound on our lot.specifctechnical data sheets' Kou can also contact technical support to discuss the recommended use time oreach er7inlmer radiochemical . our contact inormation is on the upper ri#ht.hand corner o this pa#e'

ner#y 56 releases "eta ener#y1 which can "e measured on a scintillation counter ater the

addition o scintillant1 in the orm o a scintillation coc7tail' The "eta ener#y interacts with the scintillant toproduce photons1 which are measured "y the detector'

,34I releases "oth "eta.li7e ener#y and #amma ener#y' I you only have access to a #amma

counter1 you should use a radioli#and la"eled with ,34I'

Antiserum

For optimi@ed !IA 7its1 the antiserum $"inder% is provided at a concentration chosen to #ive optimi@ed assay

cali"ration curve sensitivity' It should "e at an appropriate dilution to provide 5- to E-> "indin# o the tracerLs totalcounts when added in the a"sence o any unla"eled li#and' I the @ero standard $no analyte standard% "indin# isoutside this ran#e1 the assay results may not "e valid $see Tips and FAs section1 urther "elow%'

The antiserumLs avidity and a)nity constant properties are the primary determinants o the appropriateconcentration ran#e or the cali"ration curve and the concentrations o analyte that can "e measured in the assay'These are properties o the raw antiserum and important actors in the selection screenin# o appropriate antisera in

7it assay development'

In optimi@in# your own !IA1 assay sensitivity is optimi@ed "y usin# an appropriately titered dilution o antisera' Thisusually #ives 5-.E-> @ero standard $no analyte% "indin#1 and produces the optimum C4- cali"ration curve mid.point' This is always pre.determined in a commercially manuactured 7it'

!tandard "oncentrate

In most !IA 7its1 a solution o unla"eled cali"ration Standard Concentrate is provided alon# with a dilution

protocol to prepare a series o concentrations or a cali"ration curve' A hypothetical !IA 7it may contain a,-- n#BmG Standard Concentrate1 which is diluted as ollows to prepare a cali"ration curve appropriate or theassay

Ta"le ,' Serial dilution o cali"ration standard'

Suggested Dilution Scheme for 100 ng/mL stock Calibration

Standard

Tub

e

Concentration (pg/0.1

mL)

a -', mG $,-- G% standard M ,'= mG assay"u;er

4--

" -' mG o dilution a M -'E mG assay "u;er3--

c -' mG o dilution " M -' mG assay "u;er ,--

d -' mG o dilution c M -' mG assay "u;er 4-

e -' mG o dilution d M -' mG assay "u;er 34

-' mG o dilution e M -'E mG assay "u;er,-

# -' mG o dilution M -' mG assay "u;er 4
http://www.perkinelmer.com/coahttp://www.perkinelmer.com/coahttp://www.perkinelmer.com/coa


4/10

Suggested Dilution Scheme for 100 ng/mL stock Calibration

Standard

Tub

e

Concentration (pg/0.1

mL)

h -' mG o dilution # M -'E mG assay "u;er 3

HThis concentration represents actual mass added to assay tu"e' Dilutions " throu#h h should "e used or thestandard curve'

HHipets andBor pipet tips used to transer diluted standard must "e made o polypropylene or siliconi@ed #lass'

HHHCali"ration Standards must "e diluted resh on the day o the assay'

#recipitating Reagent

The recipitatin# !ea#ent allows the separation o "ound li#and.anti"ody comple(es rom ree li#and and

anti"ody remainin# un"ound' A solution containin# ,E> polyethylene #lycol $N E---% and -'-4> sodium a@ide in4- m0 phosphate "u;er1 p6 E'/ may "e used' Alternatively1 an anti.isotype specifc secondary anti"ody $such assheep anti.ra""it serum to precipitate ra""it anti.li#and% may "e used' Some carrier ra""it serum is #enerally

re&uired'

Other precipitatin# rea#ents include secondary anti"ody.coated Scintillation ro(imity Assay $SA% "eads or coatedma#netic particles'


5/10

Figure . &chematic for a radioimmunoassay. Radioactive antigen("tracer") is added to the antibody follo$ed by addition of unlabeled antigen (from sample or from standard). 'he antigen-antibody complees

formed are precipitated using a precipitating reagent (in the eample sho$n a secondary antibody) to separate bound and free tracer.

$%uipment re%uired

ipettors andBor pipets that accurately and precisely deliver the re&uired volumes

olypropylene test tu"es

Test tu"e rac7

8ea7ers or as7s

Porte( mi(er

Centriu#e $reri#erated1 with swin#in# "uc7et rotor%

Gi&uid scintillation counter $such as aTri.Car"Q or0icro8etaR li&uid scintillation counter% or #amma counter

$such as aWi@ard3 #amma counter% as applica"le

top

Definitions

A)nity $potency% the ti#htness with which the li#and "inds to the receptor or anti"ody "indin# site' This is usuallye(pressed as an e&uili"rium constant1 *d' The lower the *d value1 the hi#her the a)nity' This also relates to theconcentrations o unla"eled li#and that can "e measured in the competitive assay'

Specifcity or Cross.reactivity descri"es how selective an anti"ody "indin# site is or a particular li#and1 and whichstructurally.similar li#ands mi#ht interere with the assay
http://www.perkinelmer.com/Catalog/Family/ID/Tri%20Carb%20Liquid%20Scintillation%20Countershttp://www.perkinelmer.com/Catalog/Family/ID/MicroBetahttp://www.perkinelmer.com/Catalog/Family/ID/MicroBetahttp://www.perkinelmer.com/Catalog/Family/ID/WIZARD2%20Automatic%20Gamma%20Counterhttp://www.perkinelmer.com/Catalog/Family/ID/WIZARD2%20Automatic%20Gamma%20Counterhttp://www.perkinelmer.com/Catalog/Family/ID/WIZARD2%20Automatic%20Gamma%20Counterhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#tophttp://www.perkinelmer.com/CMSResources/Images/44-148812ria_main_3.jpghttp://www.perkinelmer.com/Catalog/Family/ID/Tri%20Carb%20Liquid%20Scintillation%20Countershttp://www.perkinelmer.com/Catalog/Family/ID/MicroBetahttp://www.perkinelmer.com/Catalog/Family/ID/WIZARD2%20Automatic%20Gamma%20Counterhttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#top


6/10

*d The concentration where 4-> o the receptors or anti"ody "indin# sites are occupied "y radioli#andBtracer'

IC4- Concentration o a competin# li#and that displaces hal o the radioactive li#and' This is the cali"ration curvemid.point'

top

Types of assaysSaturation assa!

In creatin# or optimi@in# your own radioimmunoassay1 a saturation e(periment is usuallyperormed' This e(periment measures "indin# e&uili"rium via titration o radioli#and $tracer%1 7eepin# the amount oreceptor or antiserum dilution constant' Kou can use saturation curves to determine 8ma( $"indin# site e(pressionlevel% and *d $"indin# a)nity o li#and.anti"ody interaction%' Choosin# a concentration o radiola"eled li#and $tracer%at a"out 4-.E-> o the saturation level #enerally #ives the correct tracer mass to use in the assay'

". #hat concentrations of radioligand should $ use for m! saturation cur%e&A' We #enerally recommend you choose 5.4 concentrations "elow the estimated *d1 and 5.4 concentrations a"ove theestimated *d' The hi#hest concentration tested should "e ten times the *d $,- ( *d%'

Fi#ure shows the #raphic representation o a saturation "indin# assay1 usin# increasin# concentrations o thetritiated radioli#and cyclopentyl.,15.dipropyl(anthine with a constant amount $concentration% o "inder' In this case1the "inder is a receptor mem"rane at ,- # per mG' In a radioimmunoassay1 the "inder would "e a specifed dilution oantiserum'

Figure *. &aturation curve sho$ing binding of +-cyclopentyl-1-dipropylanthine to its receptor. #ncreasing amounts of radioligand are added to a ed concentration of receptor. 'otalbinding, #ncreasing concentration of radioligand in absence of cold ligand. easures both specic binding to receptor as $ell as non-specicbinding. on-specic binding, #ncreasing concentration of radioligand in presence of ecess unlabeled ligand. easures binding of theradioligand to non-receptor or non antibody components. &pecic binding, 'otal minus non-specic binding. easures binding to the receptoror antibody specically.

Competition assa!

Competition assays measure the e&uili"rium "indin# o a sin#le concentration o radioli#and $tracer% in the presence ovarious concentrations o competin# unla"eled cali"ration li#and $standard% or sample o un7nown concentration'

rotocol or a hypothetical !IA

,' &uili"rate all rea#ents to room temperature and mi( "eore use'3' Ga"el duplicate tu"es or total counts1 S8 $"lan7%1 each standard1 and each sample $reer to Ta"le 3 "elow%'5' lace tu"es in a suita"le test tu"e rac7'' Incu"ate overni#ht $,E . 3 hours% at 3 . /C'4' Add , mG o cold recipitatin# !ea#ent to all tu"es e(cept total counts1 and mi('E' Incu"ate or 3- . 5- minutes at 3 . /C1 and centriu#e at 3 . /C' or 5- minutes at ,--- . 3--- ( g'
http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#tophttp://www.perkinelmer.com/CMSResources/Images/44-148814ria_saturation.jpghttp://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/radioimmunoassay.xhtml#top


7/10


8/10

Ta"le 5' !aw and processed data'

Tube 'o. C+, *%g C+, 'et *%g C+, -/0

TotalCounts

, ,=E5

3 ,44E5 ,43E5

8lan7 5 ,4

5/- 5=/

2-2Standard

4 //


9/10

Figure /. &tandard curve for 0-eto P2F1. 'he concentration of an un3no$nsample is interpolated from the curve.

top

Tips and FAQsarticularly when usin# an automated data reduction system1 it is essential that you review your raw data' Chec7 thatyour Total Count tu"es1 non.specifc "indin#1 @ero standard "indin# and cali"ration curve shape ma7e sense' I any oyour assay components are de#radin#1 you will notice "y inspectin# your data'

The area o your cali"ration curve "etween your @ero standard $no analyte standard% "indin# and your frst

cali"ration curve standard will "e "elow the sensitivity o detection' I you need to measure samples "elow this1 youwill need to concentrate them1 or'''

Another tric7 that you can try is a delayed addition assay' Kou can usually increase the sensitivity o detection

o the assay "y incu"atin# the anti"ody and the cali"ration standardsBsamples or 5. hours without the tracer' Aterthis initial incu"ation1 add the tracer1 mi( the tu"es and incu"ate overni#ht' I needed1 you could also optimi@e asecond assay with a more.sensitive "inder $anti"ody%'

Try to standardi@e your incu"ation times1 especially overni#ht incu"ations1 to prevent inter.assay variation'

6ydropho"ic li#ands will #enerally show hi#her non.specifc "indin#' Addition o Tween or Triton .,--

deter#ent may help this'

Xse hi#h purity radioli#andsBtracers $typically Y =-> pure%

Xse hi#hly selective anti"odies $with low cross.reactivity to structurally.similar compounds%'

Sta"ility . i you will need to use your radioli#and over an e(tended period o time1 sta"ility may "e a actor'

,34I.la"eled li#ands should #enerally "e used within one to two months o manuacture date' The amount o cpmLswill naturally decrease as the radioisotope decays' Tritiated li#ands should usually "e used within 5.E months omanuacture date $however1 there are e(ceptions to this%'

top

References

,' !adioimmunoassay' W'0' 6unter in 6and"oo7 o (perimental Immunolo#y.Polume , Immunochemistry1 D'0'Weir $d%1 8lac7well Scientifc u"lications1 Gondon ,=


10/10

Custom laelin! services

er7inlmer o;ers custom ,34I and 56 radiola"elin# services' I you are interested in custom services1 please contact ourcustom teamsOYOITQ Custom Ga"elin# Services
http://www.perkinelmer.com/Catalog/Category/ID/Custom%20Radiosynthesis%20Serviceshttp://www.perkinelmer.com/Catalog/Category/ID/Custom%20Radiosynthesis%20Services

Documents

Radio Immuno as Says