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British Journal of Anaesthesia 112 (2): 213–16 (2014)
doi:10.1093/bja/aet293
EDITORIALSERIESONMETHODOLOGY5
Radioimmunoassay,enzymeandnon-enzyme-based
immunoassays
R. D. Grange, J. P. Thompson and D. G. Lambert*
DepartmentofCardiovascularSciences,DivisionofAnaesthesia,CriticalCareandPainManagement,LeicesterRoyalInfirmary,
UniversityofLeicester, Leicester, UK
* E-mail: dgl3@le.ac.uk
Downloaded from https://academic.oup.com/bja/article/112/2/213/284639 by guest on 13 September 2022
The ability to quantify the amount of a specific protein in a format is therefore critical and the remainder of this article
complex sample has been a valuable addition to laboratory coversthemainformatscurrentlyavailable.
science,allowingthedevelopmentofdiagnostictests,allergen
detectioninthefoodindustry,andscreeningforimmunity.This Radioimmunoassay
is particularly important in anaesthesia, intensive care, and AnRIArequiresthefollowing:asamplecontainingtheantigen
pain research for the quantification of mediators (cytokines, of interest, a complementary antibody, and a radiolabelled
peptides, and analytes) involved in inflammation, pain, and versionoftheantigen.Thesampleantigenandantibodyarein-
otherpathways. cubatedtogether,allowingthesampleantigentobindwiththe
Immunoassaysusethehighspecificityofantibodies,along antibody. Theradiolabelledantigenisthenadded.Theradiola-
with their enormous diversity, to target specific molecules of belledantigencompeteswiththesampleantigenanddisplaces
interest and analyse their concentration in a sample. The first it fromtheantibody.Themoresampleantigenpresent,theless
1
immunoassaydevelopedwasdescribedbyYalowandBerson theradiolabelledantigenisabletobindtotheantibody.Asec-
in1959.2Theyusedradiolabelledinsulintoassesstheconcen-
trationofinsulininhumanplasma,andthusdevelopedthefirst ondantibodythatbindstheprimaryantibodycanthenbeadded,
radioimmunoassay (RIA). In 1971, Engvail and Perlman3 alongwithserumfromthespeciesoftheprimaryantibody,to
described a technique whereby antigens were immobilized causethesolutiontoflocculateandallowforseparationofthe
onamicroplatewell,incubatedwithantiserum,andthenthe primary antibody from solution. Since solution containing
concentration of antibody in the antiserum was quantified antigen–antibodycomplexismoredensethanthatcontaining
using an enzyme-linked anti-immunoglobulin antibody. This free-antigen, centrifuging this mixture allows separation, result-
method is the enzyme-linked immunosorbent assay (ELISA). inginapelletcontainingtheboundsampleantigen/radiolabelled
Enzymeimmunoassays(EIAs)areverysimilar to ELISAs, and antigen.Bymeasuringtheradioactivityofthepellet,itispossible
as such, the terms are often used interchangeably. The EIA todeterminetheamountofradiolabelledantigenthathasbound
4 to antibody, and therefore the concentration of antigen in the
wasdevelopedbyVanWeemenandSchuurs (independently sample(Fig.1).ThedrawbacksofRIArelatetotheuseofaradio-
of Engvail and Perlman) for the quantification of antigen 125
rather than antibody. For the purpose of this article, EIA and label(usually[ I])andhenceshortshelflife.Theseassaysdonot
ELISAshouldbeconsideredinterchangeable. use enzymes and thus reduces the risk of interference from the
The majority of RIA assay formats recommend sample sampleitself.
cleaning and concentration (particularly when analyte con- Enzyme-linkedimmunosorbentassay
centrationandassaysensitivityislow),althoughalargenum-
ber of ELISA assays can cope with direct use of unprocessed ThereareavarietyofELISAmethods.Theimportantvariations
plasma. The cleaning and concentration process usually aredescribedbelow(Fig.2).
involvesionexchangechromatographyfollowedbysomeform
offreezedrying/lyophilization.Wewouldrecommendusersto DirectELISA
determineifsamplecleaningisrequiredfortheiranalyte. ThisisthesimplestoftheELISAtechniques.Thesampleisfirst
Often,therearedifferencesinmeasuredanalyteconcentra- added to the microplate well and incubated. The sample will
tion when comparing RIA and ELISA. This can result from containtheantigenofinterest.Theantigenbecomesadsorbed
specificity of the antibody (e.g. the cardiovascular peptide onto the surface of the well. The wells are then washed thor-
56
urotensin II) or the fluid in which the analyte is suspended oughly,leavingonlytheabsorbedantigen.Remainingbinding
interfering with onlyonetypeofassay(e.g.theopioid-related sites on the well are then blocked. An antibody, complemen-
peptide Nociceptin/Orphanin FQ).7–11 Discordance has also tary to the antigen of interest, is then added to the wells
beendemonstratedbetweenRIAsandEIAsmeasuringcortisol where it binds to the antigen. The well is again washed. This
12 13
and carcinoembryonic antigen. The selection of assay leaves a bound antigen–antibody complex on the surface of
&TheAuthor[2014].PublishedbyOxfordUniversityPressonbehalfoftheBritishJournalofAnaesthesia.Allrightsreserved.
ForPermissions,pleaseemail:journals.permissions@oup.com
BJA Editorial
the well. The bound antibody will have attached to it an Therestoftheexperimentcannowproceedinthesameway
enzyme.Asubstrateisthenaddedwhichwillbeconvertedby asadirectoranindirectELISA.
the enzyme into a detectable product. Detection may be The clear benefit of this method is improved sensitivity. It
basedoncolour,fluorescence,orluminescence. does however come at a cost. For this method to work, two
Thismethodhastheadvantageofbeingquickerandsimpler antigen-specific antibodies are required. They need to bind to
thantheotherELISAmethods,withfewersteps,andjustone different epitopes on the antigen, and these need to be far
antibody.Itdoes,however,havesomelimitations.Incomplex enoughawayfromeachotherastonothinderthebindingof
samples,containingarangeofdifferentproteins,therewillbe one another. If a secondary antibody is used (as in indirect
a variety of proteins adsorbed onto the well that are not the ELISA),itisimportantthatthecaptureandprimaryantibodies
antigenofinterest. This proves problematic whenthe antigen are raised in different species. This is because the secondary
of interest is in low abundance as the sensitivity of the test is antibodywillberaisedagainstthespeciesoftheprimaryanti-
reduced. Another issue is that the antibody needs to have an body.Ifbothcaptureandprimaryantibodywerefromthesame
enzymeattachedtoit.Thiscostlyandtime-consumingprocess species, then the secondary antibody would bind to both and Downloaded from https://academic.oup.com/bja/article/112/2/213/284639 by guest on 13 September 2022
hastoberepeatedforeachindividualELISA,aproblemavoided notreflectdifferencesinboundantigen.
bytheothermethods.Also,conjugatingtheantibodywithan
enzymehasthepotentialtoreducetheaffinityoftheantibody CompetitiveELISA
to the antigen, and thus reduce sensitivityonce more. This methodrequirestwoligandstocompetewitheachother
for a limited number of antibody sites. One ligand will be the
IndirectELISA antigen of interest, and one will be a similar molecule that is
able to bind to the antibody, but has a variation that allows a
Samplecontainingtheantigenofinterestisadsorbedontothe furthermoleculetoexclusivelybindtoit.Thisisoftenachieved
wellsofamicroplate,followedbyblockingofremainingsiteson byaddingbiotintotheantigenofinterest.Theantigenandthe
thewell.Acomplimentaryantibody(primaryantibody)isthen biotinylatedantigenwillcompeteforthesamesiteontheanti-
added, which binds to the antigen forming a complex. This body. The signal generated by this assay will be inversely pro-
method differs from the direct method in that the antibody portional to the amountofantigeninthesample.
binding to the antigen does not have attached to it an As mentioned, biotin is often added to the competing
enzyme or any other signal-generating substance. Instead, antigen.Itisausefulmoleculesinceitissmall,andthusdoes
thepurposeofthisantibodyistoactasabridgebetweenthe notappreciablyreducetheaffinityoftheantigenfortheanti-
antigen and a secondary (enzyme-linked) antibody. This sec- body. It also binds readily and specifically to streptavidin.14
ondary antibody will have been raised in an animal different Streptavidin is a protein that is easily conjugated to a variety
fromthatoftheoriginoftheprimaryantibodyandwilltarget ofmolecules,allowingsignalgenerationfromavarietyofsour-
theFcregionoftheprimaryantibody. ces such as colour changes, chemiluminescence (immunolu-
Thesecondaryantibodyisoftenpolyclonal(originatesfrom 15
minometric assay), and fluorescence (immunofluorometric
differentBcells)andassuchwillberesponsivetodifferentepi- 16
assay). The biotin–streptavidin complex can also be used
topesontheprimaryantibody.Thisallowsmultiplesecondary asasignalamplifier.
antibodies to bind to the same primary antibody, thereby
amplifyingthesignalandincreasingthesensitivityofthetest Otherimmunoassays
(although there is still the issue of complex samples having
multipleproteins adsorbedontothesurfaceofthewell). The use of enzymes in an assay can be advantageous since
Another advantage of this method is the exclusion of the this allows for the use of a variety of substrates that can
needtoconjugatetheprimaryantibody,avoidingtheproblems generate different signals. Enzymes are, however, open to
describedabove.Secondaryantibodiescanthereforebemade interference.Forexample,horseradishperoxidaseandalkaline
commercially available at a much lower price, and with a phosphatase are the most frequently used enzymes and are
variety of signal-producing conjugates (i.e. all ELISAs using a inhibited by buffers containing sodium azide (a commonly
rabbit-derivedprimaryantibodycouldusethesameanti-rabbit used preservative) and phosphate, respectively. Endogenous
IgGsecondaryantibody). sample peroxidases and phosphates may also interfere with
theassay.
SandwichELISA Immunoassaysthatdonotrequiretheuseofenzymesand
radionuclides are now being developed. These assays include
Thedirectandindirectmethodsbothsufferfromthefactthat competition assays using fluorescent peptides, and also a
complexsampleswillreducethesensitivityoftheexperiment varietyoflabelledstreptavidincompoundsforusewithbiotiny-
duetoavarietyofproteinsadsorbingtothewell.Thesandwich latedantibodiesorpeptides.
methodovercomesthis.Anantibodycomplementarytothatof The above assay formats are heterogeneous immunoassays
theantigen(captureantibody)isfirstaddedtotheplatewhere (assaysthatrequireseparationofboundandunboundantibody/
it is adsorbed to the well. A blocking agent is added as before antigen before signal recording). Other assays, such as Enzyme
andasampleisthenadded.Onlytheantigenofinterest can multiplied immunoassay technique (EMIT)17 and Fluorescence
remain on the plate since it is able to bind to the antibody. 18
polarization immunoassays (FPIA) do not require this
214
Editorial BJA
A B Primary antibody
Sample peptide
Radiolabelled peptide
Secondary antibody
100 G
ESupernatant can then be 80
removed. The resulting 0
pellet can be measured B 60
/
for radioactivity. B
CDRadiation levels will be % 40
inversely proportional to 20 Downloaded from https://academic.oup.com/bja/article/112/2/213/284639 by guest on 13 September 2022
sample peptide levels
0
F −13 −12 −11 −10 −9
LogUII(M)
Radioactivity
Concentration
Fig 1 (A) Sample peptide is incubated with primary antibody. (B) Radiolabelled peptide is then added. It competes with sample peptide
C) Secondary antibody binds to primary antibody and causes it to precipitate out of solution. (D) Centrifugation causes the
and displaces it. (
antibody–antigencomplextoformapellet.(F)Exampleofatypicalstandardcurve.(G)Actualstandardcurveforurotensin-II(UII)whereamount
of radioactive iodine bound is expressed as B/B which is the ratio of binding at each standard concentration, B to that bound in the absence of
0
displacer, B . Analytesamplesinbiologicalspecimensshouldlieonthestraightpartofthecurve.
0
A B
Primary antibody 6.0 E
5.5
Sample peptide 5.0
Labelled peptide Log RLU4.5
C D
4.0
Secondary antibody
3.5
Antibody label −12 −11 −10 −9 −8
(Enzyme or biotin) –1
Log [TNF-a](g ml )
Fig2 Schematicshowingthedifferencesbetweendirect(A),indirect(B),sandwich(C),andcompetitive(D)EIAmethods.(E)Actualstandardcurve
forasandwichTNF-aassay.NotethewaythestandardcurveispresentedvarieswiththeRIAinFigure1,butanalytesamplesinbiologicalspeci-
mensshouldlieonthestraightpartofthecurve.RLU,relativelightunitssignalfromtheenzymereaction.
separation, and are classified as homogenous immunoassays. distinguished. Some recent British Journal of Anaesthesia RIA/
EMITrequires an enzyme-linked antigen that will compete with ELISAdataaresummarizedinTable1.
sample antigen for antibody binding. The enzyme is designed
so as to become deactivated by antibody binding. FPIA works Declarationofinterest
similarly, with fluorescein-conjugated antigens competing.
Bound and unbound fluorescein-conjugated antigens emit D.G.L. holds a consultancy with Grunenthal GmbH, but this is
fluorescence of different intensities and can therefore be not directly related to the content of this article. D.G.L. is the
215
BJA Editorial
Table1 SomeELISA(Sandwich)/RIAassayformatsusedinstudiespublishedrecentlyinBritishJournalofAnaesthesia.*Sensitivityquoted
Analyte Manufacturer Method Range Reference
HumanIL-1b R&DSystems Sandwich 3.9–250pgml21 19
HumanIL-6 R&DSystems Sandwich 3.12–300pgml21
HumanIL-8 R&DSystems Sandwich 31.2–2000pgml21
HumanIL-10 R&DSystems Sandwich 7.8–500pgml21
TNFalpha R&DSystems Sandwich 0.5–32pgml21
Neuralgrowthfactor Promega Sandwich 3.9–250pgml21 20
Heatshockprotein70 EnzoLifeSciences Sandwich 780–50000pgml21 21
Heatshockprotein90 EnzoLifeSciences Sandwich 62.5–4000pgml21
Heatshockprotein60 EnzoLifeSciences Sandwich 3.125–100ngml21
21 Downloaded from https://academic.oup.com/bja/article/112/2/213/284639 by guest on 13 September 2022
b-Endorphin* RIA 10pgtube 22
administrationdirectorandaboardmemberofBJA,andJ.P.T.is 12 RaffH,HomarPJ,BurnsEA.Comparisonoftwomethodsformeas-
aneditorandboardmemberofBJA. uringsalivarycortisol. Clin Chem2002;48:207–8
13 FleisherM,NisselbaumJS,LoftinL,SmithC,SchwartzMK.RocheRIA
and Abbott EIA carcinoembryonic antigen assays compared. Clin
Chem1984;30:200–5
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