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:

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