Radioimmunoassays

Article Outline
Overview
What do I need to run this assay?
Defnitions
Types of assays
o Saturation assay
o Competition assay
Calculations
Tips and FAs
!eferences
Custom la"elin# services
Overview
!adioimmunoassays $!IAs% use anti"odies to detect and &uantitate the amount of anti#en $analyte% in a sample' These
assays are typically very sensitive and specifc' It is possi"le to detect as low as a few pico#rams of analyte in the
e(perimental tu"e when usin# anti"odies of hi#h a)nity $*d + ,-
./
. ,-
.,,
0%' The "asic principle of radioimmunoassay
is competitive "indin#1 where a radioactive anti#en $2tracer2% competes with a non.radioactive anti#en for a f(ed
num"er of anti"ody or receptor "indin# sites' When unla"eled anti#en from standards or samples and a f(ed amount
of tracer $la"eled anti#en% are allowed to react with a constant and limitin# amount of anti"ody1 decreasin# amounts
of tracer are "ound to the anti"ody as the amount of unla"eled anti#en is increased'
In our
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I and
5
6 radioimmunoassay 7its1 separation of the anti"ody.anti#en comple(es from free anti#en is achieved
"y precipitation of the anti"ody."ound tracer with either a secondary anti"ody solution directed a#ainst the #enus or
species specifc immuno#lo"ulins of the primary anti"ody1 or "y use of polyethylene #lycol' 8oth precipitators
#enerally re&uire the presence of carrier immuno#lo"ulin' After centrifu#ation1 the supernatant containin# the un"ound
anti#en is decanted1 and the pellet containin# the anti"ody.anti#en comple( is counted in a scintillation
counter' !esults o"tained for the standards are used to construct a standard $dose.response% curve from which the
un7nowns are calculated "y interpolation'
Figure 1. Principle of a competitive binding
radioimmunoassay. Radiolabeled antigen ("tracer") added to an antibody specifc to the antigen leads to formation of an antigen-antibody
comple. !nlabeled antigen from a sample or standard solution can also bind antibody" leading to unlabeled antigen-antibody comple. #n the
radioimmunoassay" the amount of radiolabeled antigen (tracer) is held constant. #ncreasing amounts of unlabeled antigen in the sample $ill
compete $ith tracer for binding to the antibody" leading to more unlabeled antigen-antibody comple.
Fi#ure 3 illustrates how a limited amount of anti"ody "indin# sites contained in a test tu"e or microplate well can
either "ind unla"eled li#and $in this e(ample1 6epatitis 8 Surface Anti#en% or radiola"eled li#and' As the amount of
unla"eled li#and increases1 there is conse&uently less radiola"eled li#and "ound' The unla"eled li#and can come from
either a 9cali"ration standard: or the sample that you are tryin# to measure'
Figure %. a) &ample containing a high amount of
antigen. '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).
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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@e
nonspecifc anti#en 9stic7in#: to the reaction tu"es1 -'5> "ovine serum al"umin may "e added' Other additives
may include ,- m0 ADTA andBor -'4> Tween.3-'
Radiolabeled
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I- or
5
H- tracer. Two terms must be understood when optimizing an RIA tracer concentration
Specifc ActivityC This is the amount of radioactivity that is present on the la"eled anti#en $li#and%' The units
are usually CiBmmol or DCiBD#' 8y 7nowin# this value1 the mass of diluted tracer per unit volume in the assay is
7nown' The ideal mass concentration of tracer in an assay is set at sli#htly "elow the saturatin# concentration for
the antiserum dilution used in the assay' Ideally1 5-.E-> of the la"eled li#and $@ero standard "indin#% should "e
"ound in the a"sence of unla"eled li#and at the appropriate tracer mass and antiserum dilution' This provides
optimum assay sensitivity' !adioli#ands with a hi#h specifc activity are well.suited for li#and "indin# assays'
Specifc activity indicates how much radioactivity there is per molecule of li#and1 and is usually #iven in units of
Curies per millimole of li#and' Several of our
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I.la"eled li#ands are o;ered at ma(imum specifc activity $33--
CiBmmol if one
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I la"elin# site is availa"le1 FF-- CiBmmol if two ,34I la"elin# sites are availa"le1 etc'%' This indicates
that virtually every molecule of li#and provided in the stoc7 vial is radiola"eled' For tritiated li#ands $56 li#ands%1
you should ideally pic7 a li#and that has a specifc activity a"ove 3- CiBmmol' The ma(imum theoretical specifc
activity per tritium is 3= CiBmmol $Curies per millimole of tritium%' Specifc activities a"ove this value indicate that on
avera#e1 each molecule of li#and has at least one tritium'
Activity ConcentrationC This is the concentration1 usually in DCiBmG1 of radioactivity per unit volume of solvent
or diluent' It has little to do with the actual optimi@ation of tracer mass1 "ut serves as a re&uired measurement of
the correct amount of counts added to the assay' This is important for the user to 7now "ecause the calculation of
non.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 perform the
recommended dilution in assay "u;er and use the appropriate volume per assay tu"e'
HHIipets andBor pipet tips used to transfer the tracer solution must "e of polypropylene or siliconi@ed #lass' Do not
use those made of unsiliconi@ed #lass1 as there may "e stic7in# issues'
There are an additional few factors to 7eep in mind when selectin# a tracer for your assayC
?on.specifc "indin#C 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#' If the
stoc7 radiochemical is pac7a#ed in a silani@ed vial $refer to the tech data sheet%1 this may indicate the li#and is
somewhat hydropho"ic
6i#h purityC Ideally1 the tracer should have a radiochemical purity a"ove =->' !adiochemical
purity decreases over time1 and the actual rate of this de#radation accelerates over time' !adiochemical purity
and de#radation rates for our radiochemicals can "e found on each lot.specifc technical data sheet'
6i#h selectivityC The more selective the tracer is for 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"ilityC If you will need to use your radiola"eled tracer over an e(tended period of time1
sta"ility may "e a factor for you'
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I.la"eled li#ands should #enerally "e used within one to two months of
the manufacture date' Tritiated li#ands should usually "e used within 5.E months of manufacture dateJ
however1 there are e(ceptions to this' De#radation rates and manufacturin# dates can "e found on our lot.
specifc technical data sheets' Kou can also contact technical support to discuss the recommended use time for
each Ier7inAlmer radiochemical . our contact information is on the upper ri#ht.hand corner of this pa#e'
Aner#yC
5
6 releases "eta ener#y1 which can "e measured on a scintillation counter after the
addition of scintillant1 in the form of a scintillation coc7tail' The "eta ener#y interacts with the scintillant to
produce photons1 which are measured "y the detector'

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I releases "oth "eta.li7e ener#y and #amma ener#y' If you only have access to a #amma
counter1 you should use a radioli#and la"eled with
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I'
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# of the tracerLs total
counts when added in the a"sence of any unla"eled li#and' If the @ero standard $no analyte standard% "indin# is
outside this ran#e1 the assay results may not "e valid $see Tips and FAs section1 further "elow%'
The antiserumLs avidity and a)nity constant properties are the primary determinants of the appropriate
concentration ran#e for the cali"ration curve and the concentrations of analyte that can "e measured in the assay'
These are properties of the raw antiserum and important factors in the selection screenin# of appropriate antisera in
7it assay development'
In optimi@in# your own !IA1 assay sensitivity is optimi@ed "y usin# an appropriately titered dilution of antisera' This
usually #ives 5-.E-> @ero standard $no analyte% "indin#1 and produces the optimum AC4- cali"ration curve mid.
point' This is always pre.determined in a commercially manufactured 7it'
!tandard "oncentrate
In most !IA 7its1 a solution of unla"eled cali"ration Standard Concentrate is provided alon# with a dilution
protocol to prepare a series of concentrations for a cali"ration curve' A hypothetical !IA 7it may contain a
,-- n#BmG Standard Concentrate1 which is diluted as follows to prepare a cali"ration curve appropriate for the
assayC
Ta"le ,' Serial dilution of cali"ration standard'
Suggested Dilution Scheme for 100 ng/mL stock Calibration
Standard
Tub
e
Concentration (pg/0.1
mL)
a -', mG $,-- DG% standard M ,'= mG assay
"u;er
4--
" -'F mG of dilution a M -'E mG assay "u;er 3--
c -'F mG of dilution " M -'F mG assay "u;er ,--
d -'F mG of dilution c M -'F mG assay "u;er 4-
e -'F mG of dilution d M -'F mG assay "u;er 34
f -'F mG of dilution e M -'E mG assay "u;er ,-
# -'F mG of dilution f M -'F mG assay "u;er 4
Suggested Dilution Scheme for 100 ng/mL stock Calibration
Standard
Tub
e
Concentration (pg/0.1
mL)
h -'F mG of 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 for the
standard curve'
HHIipets andBor pipet tips used to transfer diluted standard must "e made of polypropylene or siliconi@ed #lass'
HHHCali"ration Standards must "e diluted fresh on the day of the assay'
#recipitating Reagent
The Irecipitatin# !ea#ent allows the separation of "ound li#and.anti"ody comple(es from free li#and and
anti"ody remainin# un"ound' A solution containin# ,E> polyethylene #lycol $IAN E---% and -'-4> sodium a@ide in
4- m0 phosphate "u;er1 p6 E'/ may "e used' Alternatively1 an anti.isotype specifc secondary anti"ody $such as
sheep 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 Iro(imity Assay $SIA% "eads or coated
ma#netic particles'
Figure ). &chematic for a radioimmunoassay. Radioactive antigen
("tracer") is added to the antibody" follo$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
Iipettors andBor pipets that accurately and precisely deliver the re&uired volumes
Iolypropylene test tu"es
Test tu"e rac7
8ea7ers or Oas7s
Porte( mi(er
Centrifu#e $refri#erated1 with swin#in# "uc7et rotor%
Gi&uid scintillation counter $such as a Tri.Car"Q or 0icro8etaR li&uid scintillation counter% or #amma counter
$such as aWi@ard
3
#amma counter% as applica"le
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Definitions
A)nity $potency%C the ti#htness with which the li#and "inds to the receptor or anti"ody "indin# site' This is usually
e(pressed as an e&uili"rium constant1 *d' The lower the *d value1 the hi#her the a)nity' This also relates to the
concentrations of unla"eled li#and that can "e measured in the competitive assay'
Specifcity or Cross.reactivityC descri"es how selective an anti"ody "indin# site is for a particular li#and1 and which
structurally.similar li#ands mi#ht interfere with the assay
*dC The concentration where 4-> of the receptors or anti"ody "indin# sites are occupied "y radioli#andBtracer'
IC4-C Concentration of a competin# li#and that displaces half of the radioactive li#and' This is the cali"ration curve
mid.point'
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Types of assays
Saturation assa!
In creatin# or optimi@in# your own radioimmunoassay1 a saturation e(periment is usually
performed' This e(periment measures "indin# e&uili"rium via titration of radioli#and $tracer%1 7eepin# the amount of
receptor or antiserum dilution constant' Kou can use saturation curves to determine 8ma( $"indin# site e(pression
level% and *d $"indin# a)nity of li#and.anti"ody interaction%' Choosin# a concentration of radiola"eled li#and $tracer%
at a"out 4-.E-> of 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 the
estimated *d' The hi#hest concentration tested should "e ten times the *d $,- ( *d%'
Fi#ure F shows the #raphic representation of a saturation "indin# assay1 usin# increasin# concentrations of the
tritiated radioli#and cyclopentyl.,15.dipropyl(anthine with a constant amount $concentration% of "inder' In this case1
the "inder is a receptor mem"rane at ,- D# per mG' In a radioimmunoassay1 the "inder would "e a specifed dilution of
antiserum'
Figure *. &aturation curve sho$ing binding of )+-
cyclopentyl-1")-dipropylanthine to its receptor. #ncreasing amounts of radioligand are added to a fed concentration of receptor. 'otal
binding, #ncreasing concentration of radioligand in absence of cold ligand. -easures both specifc binding to receptor" as $ell as non-specifc
binding. .on-specifc binding, #ncreasing concentration of radioligand in presence of ecess unlabeled ligand. -easures binding of the
radioligand to non-receptor or non antibody components. &pecifc binding, 'otal minus non-specifc binding. -easures binding to the receptor
or antibody" specifcally.
Competition assa!
Competition assays measure the e&uili"rium "indin# of a sin#le concentration of radioli#and $tracer% in the presence of
various concentrations of competin# unla"eled cali"ration li#and $standard% or sample of un7nown concentration'
Irotocol for a hypothetical !IA
,' A&uili"rate all rea#ents to room temperature and mi( "efore use'
3' Ga"el duplicate tu"es for total counts1 ?S8 $"lan7%1 each standard1 and each sample $refer to Ta"le 3 "elow%'
5' Ilace tu"es in a suita"le test tu"e rac7'
F' Incu"ate overni#ht $,E . 3F hours% at 3 . /SC'
4' Add , mG of cold Irecipitatin# !ea#ent to all tu"es e(cept total counts1 and mi('
E' Incu"ate for 3- . 5- minutes at 3 . /SC1 and centrifu#e at 3 . /TC' for 5- minutes at ,--- . 3--- ( g'
<' Decant the supernatants from all tu"es $e(cept total count tu"es% into an appropriate radioactive li&uid waste
tray'
/' 8lot the li&uid from the rims of the assay tu"es on a"sor"ent paper mats for U , minute'
=' Count the radioactivity remainin# in the assay tu"es $includin# the Total Count tu"es%'

Ta"le 3' Tu"es for radioimmunoassay protocol' All volumes are in microliters $DG%'
Tube 'o. (u)er Standard Samples Tracer *ntibod!
Total
Counts
,.3 ... ... ... ,-- ...
8lan7 5.F 3-- ... ... ,-- ...
2-2
Standard
4.E ,-- ... ... ,-- ,--
Standards <.3- ... ,-- ... ,-- ,--
Samples 3,1331 etc' ... ... ,-- ,-- ,--

". #hat concentration of radioligand (tracer) should be selected for competiti%e binding&
A' The radioli#and $tracer% is used at a low concentration1 usually at or "elow its *d value' If the specifc activity is low1
concentrations a"ove the *d value can "e used1 thou#h the concentration must never "e at or hi#her than saturatin#
concentrations'
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Calculations
After countin# is complete1 the concentration of analyte in the samples is determined "y interpolation from a standard
curve' The followin# method is su##ested $refer to Ta"le 5 for sample calculations%'
A' Avera#e the counts for each set of tu"e replicates'
8' Calculate the avera#e ?AT counts for all standards $includin# the Vero Standard% and samples "y su"tractin# from
each the avera#e non.specifc "indin# counts'
C' Determine the normali@ed percent "ound $> 8B8-% for each standard and sample as followsC
D' Ilot > 8B8- for each standard versus the correspondin# amounts of unla"eled li#and cali"rator added in
pico#rams' See Fi#ure 4 for a typical standard curve usin# the standard protocol'
A' Determine the amount of analyte in each sample "y interpolation from the standard curve' 8ecause the standard
curve is e(pressed as 2pico#rams added21 sample values must then "e corrected for any dilutions to determine the
ori#inal concentration in the sample'
?OTAC Any samples with concentrations which are a"ove the ran#e of the standard curve must "e diluted with assay
"u;er and re.assayed' The values o"tained are then multiplied "y the appropriate dilution factor' Palues o"tained that
are lower than the frst cali"ration standard are suspect'
Ta"le 5' !aw and processed data'
Tube 'o. C+, *%g C+, 'et *%g C+, -(/(0
Total
Counts
, ,F=E5
3 ,44E5 ,43E5
8lan7 5 F,4
F 5/- 5=/
2-2
Standard
4 //<=
E /E<5 /<<E /5</ ,--
3 p# < <<-,
/ <=5= </3- <F33 //'E
4 p# = E/<5
,- E//E E//- EF/3 <<'F
,- p# ,, 4E53
,3 4=-- 4<EE 45E/ EF',
34 p# ,5 F3,E
,F F34E F35E 5/5/ F4'/
4- p# ,4 5-/3
,E 3==4 5-5= 3EF, 5,'4
,-- p# ,< 334<
,/ 3--5 3,5- ,<53 3-'<
3-- p# ,= ,F=,
3- ,EE, ,4<E ,,</ ,F',
Sample 3, 4/3=
33 4=,= 4/<F 4F<E E4'F
Figure /. &tandard curve for 0-1eto P2F1. 'he concentration of an un3no$n
sample is interpolated from the curve.
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Tips and FAQs
Iarticularly when usin# an automated data reduction system1 it is essential that you review your raw data' Chec7 that
your Total Count tu"es1 non.specifc "indin#1 @ero standard "indin# and cali"ration curve shape ma7e sense' If any of
your assay components are de#radin#1 you will notice "y inspectin# your data'
The area of your cali"ration curve "etween your @ero standard $no analyte standard% "indin# and your frst
cali"ration curve standard will "e "elow the sensitivity of detection' If you need to measure samples "elow this1 you
will need to concentrate them1 or'''
Another tric7 that you can try is a delayed addition assay' Kou can usually increase the sensitivity of detection
of the assay "y incu"atin# the anti"ody and the cali"ration standardsBsamples for 5.F hours without the tracer' After
this initial incu"ation1 add the tracer1 mi( the tu"es and incu"ate overni#ht' If needed1 you could also optimi@e a
second 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 of Tween or Triton W.,--
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 . if you will need to use your radioli#and over an e(tended period of time1 sta"ility may "e a factor'
,34I.la"eled li#ands should #enerally "e used within one to two months of manufacture date' The amount of cpmLs
will naturally decrease as the radioisotope decays' Tritiated li#ands should usually "e used within 5.E months of
manufacture date $however1 there are e(ceptions to this%'
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References
,' !adioimmunoassay' W'0' 6unter in 6and"oo7 of A(perimental Immunolo#y.Polume , Immunochemistry1 D'0'
Weir $Ad%1 8lac7well Scientifc Iu"lications1 Gondon ,=</'
3' !adioimmunoassay' *'A' *ir7ham Z W' 0' 6unter1 Williams Z Wil7ins Co'1 8altimore1 0D ,=<,
5' Iersonal Communications. S' !ichard 6arris Ih'D1 Dupont.?A? 8iomedical Iroducts'
F' Iersonal Communications. David 6andfeld and Scott *eohane1 Ier7in Almer Gife Z Analytical Sciences'
4' Immunolo#y . Dr [anis *u"y . W'6' Freeman Z Co'1 ?K 3--<
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Custom laelin! services
Ier7inAlmer o;ers custom
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I and
5
6 radiola"elin# services' If you are interested in custom services1 please contact our
custom teamsC
O?YIOI?TQ Custom Ga"elin# Services