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Antibody Specificity:What's the problem?The Antibody Society Webcast series – Antibody Validation #1
Andreas Plückthun University of Zurich
Antibodies are known to be specific.So how can there be a problem?The main reason: • They have not been checked
for specificity • Specificity cannot be assumed,
but must be experimentally verified
What causes non-specific binding, and absence of specific binding? 1. Protein surfaces always bind several things 2. Antigens can be in various conformations, which present different surfaces 3. Composition of an antibody solution may not be what you think it is
Fundamental properties of proteins:They bind one another!
• Proteins(antigens,antibodies)havetheintrinsicpropertyofinteractingwithotherproteins• throughadventitioushydrophobicpatches• thoughadventitiousresiduesthatcanmakehydrogenbonds
• Sinceantibodiesareproteins,theycross-reactwithproteins(antigens)unrelatedtotheirantigens–albeitatverydifferentaffinities
Types of antibodies
Polyclonalantibodies:
• Popular,becausetheyarecheap• takendirectlyfromserum
• Andcangivestrongsignals• theytakeadvantageofmanyepitopes• theycanbindbivalentlyinmanyorientations
Types of antibodies
PolyclonalantibodiesBut:• Therearealwaysantibodiesintheantiserathatcrossreactwithothercomponents• Thecompositionoftwoantiserawillneverbethesame• Itisimpossibletoreproduceresultsfrompolyclonalsera
Types of antibodies
MonoclonalantibodiesPopular,becausetheyarebelievedtobeautomaticallysuper-specific
Types of antibodies
MonoclonalantibodiesBut:• Theycanalsocrossreactwithotherproteins• Theymaydetectotherproteinsbetterthanthedesiredtarget• A“monoclonalantibody”isnotnecessarilymonoclonal!
Types of antibodies
MonoclonalantibodiesBut• Amonoclonalantibodycanalsocrossreactwithotherproteins
Theexpectedcase:relatedproteins"Legitimatecrossreactivity"
antibody antigens
desired
undesired
closelyrelatedprotein
Types of antibodies
MonoclonalantibodiesBut• Amonoclonalantibodycanalsocrossreactwithotherproteins• Ifnotcheckedproperly,itmaydetectotherproteinsbetterthanthedesiredone
The(perhaps)unexpectedcase:unrelatedproteins-maybeunfolded,"sticky"-mayhaveonlyfewepitopes"Illegitimatecrossreactivity"
antibody antigens
Types of antibodies
MonoclonalantibodiesBut• Amonoclonalantibodycanalsocrossreactwithotherproteins• Ifnotcheckedproperly,itmaydetectotherproteinsbetterthanthedesiredone
Theantibodymayevenadapttoothertargets!"Illegitimatecrossreactivity"
antibody antigens
Types of antibodies
MonoclonalantibodiesBut• Amonoclonalantibodycanalsocrossreactwithotherproteins• Ifnotcheckedproperly,itmaydetectotherproteinsbetterthanthedesiredone• Amonoclonalantibodymaynotevenbemonoclonal
Frequently(onethird!):-expressionofmorethanonealleleinB-cell
-fusionofmorethanoneB-cell
-additionallightchainsfrommyelomafusionpartner
Types of antibodies
MonoclonalantibodiesYetanotherproblem:• Aslongasthesequenceoftheantibodyhasnotbedetermined,youcannotknowwhethertwoantibodiesarethesame• Manufacturersselltoeachother(sameantibody,differentlabel)
• Manufacturersproduceanewlot,maybedifferentcomposition
• Itmaybeimpossibletoreproduceanexperiment
Frequently(onethird!):-expressionofmorethanonealleleinB-cell
-fusionofmorethanoneB-cell
-additionallightchainsfrommyelomafusionpartner
Types of antibodies
RecombinantantibodiesThesequenceisknown.Itcanbereproducedforever,theantibodyis"immortal"Ofcourse,qualitycontrolstillhastobedoneasforeveryantibody!
➔ Bymostexperts,recombinanttechnologiesareseenasthefuture
Immunization
DNAisolation SyntheticDNAlibrary
Displaytechnologies
Selectionforspecificity
Qualitycontrol
Sequencedetermination
Types of "antibodies"
RecombinantaffinityreagentsTheantibodyitselfhasbecomedispensableAffinityreagentscanbeusedthataremuchmorestablethanantibodies➔ Othernon-antibodyscaffoldsThesecanbeproducedmuchmorecheaply➔ Bymostexperts,recombinanttechnologiesareseenasthefuture
SyntheticDNAlibrary
Displaytechnologies
Selectionforspecificity
Qualitycontrol
Sequencedetermination
Types of antigens
Denatured(unfoldedproteins)• usuallyexposehydrophobicresidues,becomemore"sticky"• usuallyneedtobekeptinsolutionbydetergent(SDS),ordenaturant(urea,GdnHCl)
Foldedproteins• usuallythecellularstate• usuallymoresoluble
Types of antigens
Denatured(unfoldedproteins)• afterSDS-Gelelectrophoresis(Westernblots)• afterproteolyticdigestion• aftertissuefixation(antigen"retrieval"withamicrowaveoven!)
Foldedproteins• incellextracts(pull-downassays)• onthecellsurface(FACSexperiments)
Types of antigens
• Manyconditionscandenatureaprotein:• antibodiesthatrecognizethenativestatenolongerbind
• Heat,shaking(=foam),lossofligands,lossofmetals,lossofsubunits,...
Types of antigens
Antibodiescanrecognizelinearepitopes,whichwillonlybeaccessibleinadenaturedprotein,orinpeptidedigest • residuesthatarecloseinsequencebutmaybehiddenininterior
Antibodiescanrecognizeconformationalepitopes,whichwillonlybeaccessibleinthefoldedprotein• residuesthataretypicallyonthesurface,butfarapartinsequence
Types of antigens
Mostantibodiescanonlyrecognizeeitherthefoldedortheunfoldedstate!
Types of antigens
Mostantibodiescanonlyrecognizeeitherthefoldedortheunfoldedstate!Mostantibodiescanthusonlybeusedonlyfor• eitherWesternblots,IHC(unfoldedstaterecognition)• orFACS,pull-downs(nativestaterecognition)
Types of antigens
Mostantibodiescanonlyrecognizeeitherthefoldedortheunfoldedstate!
folded
Types of antigens
Mostantibodiescanonlyrecognizeeitherthefoldedortheunfoldedstate!
unfolded
Types of antigens
Mostantibodiescanonlyrecognizeeitherthefoldedortheunfoldedstate! ratherflat groove-like pocket
(orpeptidesidechain)
Types of antigens
Quality-controllingantibodiesisbydefinitionapplicationspecific!• Youmustcheckantigenrecognitioninthestateoftheantigenthatwillbeusedlater• Cross-reactivitywillalsodependoncontext:• otherdenaturedproteins• othercellcomponents?• non-proteinscontaminants?
Types of antigens
WhataboutELISA:• Inordertobindtopolystyrene,atleastpartoftheproteinmustdenature!• Smallproteinswillalmostcertainlydenature• Mostpeptideswillnotevenbind(canbebiotinylated)• Largeproteins:often,onlyonedomaindenatures,therestremainsfolded.(AlsotrueformanyIgGsthemselves)
Types of antigens
WhataboutImmunohistochemistry:• Antigensaretypicallycrosslinked,epitopesareblocked• Antigen"retrieval"(heat)denaturestheantigen• OnlyasmallsubsetofepitopesissuitableforIHC• Itisstillverydifficulttomimicthe"IHCconformation"invitro,andthustotestitoutsideanIHCexperiment.
Types of antigens
Arethereantibodiescanworkbothinseveralapplications?• Yes,butonlyiftheyaremadeagainstapieceoftheproteinwhichisalwaysunfolded,e.g.termini(tails)receptors
Types of antigens
Arethereantibodiescanworkbothinseveralapplications?• Yes,polyclonalantibodies• BUT:theycomewiththeveryhighpriceofcross-reactivitiesalmostimpossibletocontrol.
Summary
1. Cross-reactivityofantibodiesistobeexpected.Therefore,itmustbechecked
2. Monoclonalantibodiesarenotspecificbydefinition.Theymustbechecked
3. Cross-reactivityisapplication-specific4. Recombinantantibodiesaredefined,identifiableand
distinguishablebytheirsequence–unlikeconventionalmonoclonalantibodies,whosesequenceisnotknown. Butrecombinantantibodiesmustundergothesamechecksforcross-reactivity
Antibody Specificity:What's the problem?The Antibody Society Webcast series – Antibody Validation #1
Andreas Plückthun
University of Zürich
Next Webcast in Antibody Validation: a 9-part series1. Andreas Pluckthun : Antibody Specificity: What's the problem?
2. Glenn Begley : Antibodies and the reproducibility crisis in biological science Cecilia Williams : The Erß story – is your antibody like this? 3. Jan Voskuil : Beware the supplier OEM Andy Chalmers : Finding antibodies in the Antibody Databases 4. Anita Bardowski : Which antibody are you looking for? The RRID Jan Voskuil : Points to note on the supplier datasheets 5. Giovanna Roncador: : Correct positive and negative controls in validation 6. Aldrin Gomes : Standard technology: “even” Western blots are non-trivial Jim Trimmer : IHC issues in brain sciences 7. Travis Hardcastle : Cell KO technology Alejandra Solache : Validating Antibodies with KO technology 8. Mike Taussig : Validating antibodies using array technologies Fridjhof Lund-Johansen : Mass spectroscopy for mass validation 9. Andrew Bradbury : Why publish sequences? Andreas Pluckthun : What are the coming alternatives ?
Validation of Commercial tool Antibodies
Antibody Specificity: What's the problem? The Antibody Society Webcast series – Antibody Validation #1
Presented by Andreas Plückthun Produced and Directed by Simon L. Goodman Production Manager Fran Breden Writen by Simon Goodman https://www.antibodysociety.org/
Validation of Commercial Tool Antibodies An Antibody Society Webcast series https://www.antibodysociety.org/
Administrative Support: Dr. Fran Breden and Dr. Mini Muralidharan
Executive Director: Dr. Jan Reichert
This series would be impossible without the generous financial support of our Corporate Sponsors:
Contact us at [email protected] or +1 (508) 808-8311 to become a corporate sponsor!