Antibody Testing for COVID - The Infectious Myth€¦ · Antibodies are our body’s immune system reaction to viral proteins, known as antigens. Antibody tests incorporate antigens,

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Antibody Testing for COVID-19 DavidCrowe

[email protected]

May13,2020

Itisnowtimeforadiscussionofantibodytesting.Manypeoplenowwanttoknowhowmanyhavebeensilentlyinfectedinthegeneralpopulation,howmanyareimmune,andhowthisaffectsthefatalityrate.Thisrequiresantibodytestingandthereisatleastasmuchinterestinthisnow,astherehasbeenintheCOVID-19RT-PCRRNAtestingthatisusedtodeclaresomeoneinfected.

Executive Summary

ApositiveRT-PCRtestisusedtotellpeoplethattheyhaveCOVID-19RNAandaredeemedinfectedandinfectious,despitethetechnology’snumerousflawsandknownfalsepositives.AntibodytestsarenowbeingusedundertheassumptionthatsomeonewhoispositiveforantibodiesforCOVID-19haspreviouslybeeninfectedand,iftheyhaverecoveredfromsymptoms,isnowimmune.Antibodiesareourbody’simmunesystemreactiontoviralproteins,knownasantigens.Antibodytestsincorporateantigens,andachemicalthatallowstheintensityofthereactiontobemeasuredusinglight.Ideallyantigenswouldcomefrompurevirus,butCOVID-19virushasneverbeenpurified,thusantigensarecreatedartificiallyfromproteinsbasedonportionsofthe30,000baseRNAgenomethatisbelievedtocomefromthevirus.ThemajorantibodytypesthatarelookedforareIgM,believedtobeagenericinfectionfightingantibodythatarisesaboutaweekorsoafterinfection,andIgG,believedtobemorespecific,andbelievedbysometotakelongerforthebodytocreate.Aftertheinfectionisresolved,IgMantibodiesarebelievedtograduallydisappear,whileIgGremain,providingongoingimmunity.Unfortunately,thisidealizedpictureisnotsupportedbytheavailableevidence,eitherbecausetheevidencedoesnotexist,isinsufficient,orbecauseitdirectlycontradictsthemodel.Positiveantibodytestsshouldbeimpossiblebeforethepersonisfirstinfected(RNApositive).Yet,oldbloodsamples(2019orbefore)havetestedpositiveinsignificantnumbers.Almost14%ofsavedbloodfromolddonationstestedpositiveinaDutchstudy,andinthevalidationoftheCellexandChembiotests,4.4%and3.6%ofoldsampleswerepositive.Theidealizedantibodymodelisbasedonthedateofinfectionasthestartingpoint,butthisdateisneverknownwithcertainty.EvenwhensomeonecameintocontactwithaCOVID-19RNApositivepersononacertaindatethatisnotaguaranteethatthiswasthedateofinfection,giventhat,priortothelockdown,peoplecouldapparentlybeinfectedwhileplayinginthepark,eatingatarestaurant,walking

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downthestreet,attendingaconcert,orparticipatinginanyothernowbannedactivity.Whenantibodysurveysareperformed,thevastmajorityofpeoplewhotestpositivehadnoideathattheyhadpreviouslybeeninfected,andcannotpossiblybesureaboutthedate.Thus,theincubationperiodforthevirusisimpossibletodetermineaccurately,aswellastherangeofdaysafterinfectionthatIgMandIgGstarttodevelop.Thismakesanaccurateantibodymodelimpossibletoconstructbasedoncurrentlyavailabledata,despitenumerousbeautifulgraphsshowingthismodelinidealizedform.

Simplemodelsthatillustratethetimingofantibodiesshowthequantity(titer)risingsmoothlyand,forIgM,eventuallypeakinganddecliningsmoothly.Yetmanystudieshavefoundnegativeteststhroughoutthesymptomaticperiod.AtestdevelopedbytheWadsworthCentreinNewYorkfound40%ofsamplesnegativeforantibodies11-15daysaftersymptomsstarted,andevenmorebetween16-20days.Thisindicatesthatantibodiesmaycomeandgorandomlyandnotbehaveinasmoothandpredictablefashion.Notestdocumentation,antibodysurveysorscientificstudiesshowedthedisappearanceofIgMantibodies,predictedbythemodel,perhapsbecauseitdoesnothappen,orittakesmorethan30days,themaximumexamined.Thismightnotbeterriblyimportantinpractice,butitisanotherindicationthatthebeautifulmodelsshownintheformofgraphsaresimplistic,ifnotoutrightwrong.OtherproblemswithantibodytestsincludeasignificantnumberofsamplestestingantibodypositivefrompeoplewhowereCOVID-19RNAnegative(althoughsomehad‘COVID-like’symptoms),withnoevidencethatthepersonwaseverinfected.InoneChinesestudythepositiverateonpresumablyneverinfectedpeoplewas25%.Antibodytests,likemostinfectiousdiseasetests,areoftenreportedas‘Positive’or‘Negative’,buttheresultsarereallywhethertheintensityofacolorchangeinthetestkitwasaboveorbelowanarbitrarynumber.Thereliabilityofthiswascalledintoquestion,inadvertently,byonetestmanufacturer,whoshowedthatcontinuallydilutingsamples50:50didnotresultinahalvingofthecolorchangeateachstep.Insomecases,lessmaterialresultedinsignificantlymoreintensecolorchanges.Researchershavetriedtoconnecttheantibodytiter(inreality,thisisjustthecolorchangeintensity)withtheseverityofsymptoms,buttwoChinesepapersthatstudiedthishadtoadmitthattherewasnodifferencebetweenmildlyandseverelysymptomaticpeopleinthequantityofantibodies,norbetweenthosewithorwithoutpre-existingconditions,norinthedurationofsymptoms.Testmanufacturersalwaysruntheirtestonbloodsamplesfrompeoplewithunrelatedmedicalconditionsasacheck.Eventhoughonlyasmallnumberofsampleswereexamined,forasmallnumberofconditions,differentmanufacturersfoundasignificantpercentageofsamplespositiveforCOVID-19antibodies,thatwereknownnottohaveCOVID-19,butinsteadcontainedotherviruses,bacteriaormycoplasma,orwerefrompeoplewithauto-immuneconditions,indicatingthattheantibodiesarenotspecific.Forexample,10%ofHepatitisBsampleswerepositive,

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33%ofRespiratorySynctitiaVirus,10%ofauto-antibodiesand17%ofStreptococcus.AlargenumberofpopulationsurveyshavebeencompiledbyDeanBeelerandtheyrevealawiderangeofpercentagesofpopulationsantibodypositive,fromlessthan1%inmanycasesto32%inapoorpartofBoston.Thisisgenerallyseenasanindicationofhowfarthroughthepopulationthatthevirushasrampaged.Oneflawofmostofthesesurveysisthatthepopulationischosennon-randomly,anddoesnotrepresentthegeneralpopulation.Thegroupmaybeahouseholdsurvey,volunteers,highschoolstudentsandstaff,healthcareworkers,blooddonors,orpeoplegoingforbloodtestsatalab.Butafarbiggerproblemisthatthenumberproducedisimpossibletovalidate.When1.5%ofSantaClaravolunteerstestedpositive,itwasassumedthatthatwastruth.This‘truth’assertsthatallofthesepeoplewereRNA-positiveatsomepointintherecentpast.Butthereisabsolutelynoevidenceforthis.The‘truth’assumesthatallthepeoplewerenegativeforCOVID-19antibodiespriortotheassumedperiodofRNA-positivity.Butthereisabsolutelynoevidenceforthis.

Itassumesthatthe98.5%whotestednegativewereneverRNA-positive.Butthereisabsolutelynoevidenceforthis.Itassumesthatthe98.5%neverhadtheantibodiesbeinglookedforbefore.Butthereisabsolutelynoevidenceforthis.IcouldassertthattherealfractionpositiveinSantaClarawas98.5%,not1.5%,andthereisnolessevidenceformyassertionthanfortheresultsfromantibodytesting.

Thesesurveysoftenaskifpeoplewhotestedantibodypositivehad‘COVID-like’symptomsinthelastfewweeksormonths(andmostsaythattheydidnot).Butthesesymptoms(fever,cough,lossofsmellortaste,fatigue)aresogenericthattheyareabsolutelynotevidencethatthepeoplewerepreviouslyCOVID-19RNApositive.OnesolutionwouldbeatimeseriessurveyofalargenumberofpeoplecurrentlynegativeonbothRNAandantibodytests(uninfectedandneverinfected).Everyfewdaysthesepeoplewouldgiveadropofbloodandanasalswab.SomewouldbecomeRNApositive,andthencouldbeexaminedmorefrequentlyfortheexactpatternofantibodydevelopment,throughtothedisappearanceofIgMantibodies.Thisexperimentwouldbetimeconsuming,intrusive,inefficient(asmostpeoplemayneverbecomeinfected)andexpensive.ButconsideringthevastsumsofmoneyspentonCOVID-19research,quarantiningandtreatment,andtheevenmoretremendoussumsofmoneylostbyahobbledeconomy,andtheassertionofourpoliticiansthattheyfollowthescience(nottheheadlemming),thiswouldsurelybeworthwhile.Antibodytestsmightbefatallyflawed,buttheycanbeusedinhighlydestructiveways.Ifthenumberofpeoplewhoareantibodypositiveremainsbelowthelevelof‘herdimmunity’(90%orso)itwillbeanexcusetopromoteorevenmandatevaccination,afteravaccineisrushedontothemarket.Antibodytestscouldalsobeusedtoindefinitelyquarantinepeoplewhodonottestpositive,assertingthattheyareatdangerofbecominginfected,andthenspreadingittoothers.Theycouldbe

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usedtoseparatefamilies,arguingthatthechildrenmustbeputinfosterhomesbecausetheparentsareatriskofaninfectionatanytime.FaultytestshavebeenusedtoindefinitelyquarantineChinesecitizens.Butnow,dowehavemorecivilrightsintheUK,UnitedStates,Canadaorothermodern,oncedemocraticcountries?

Wehavebeenherebefore.ABBCstoryfrom2008,“LifeSentence”,alwaysmakesmecry.Startingin1907nearly50womenwerelockedinanasylumwithintheLongGroveinsaneasyluminSurreybecausetheyweredeemedcarriersoftyphoid.Theyweresaneandhealthywhentheyentered,butmostweredrivenmadbythesolitaryconfinement,byhumiliationsliketoiletsthatflushedboilingwater,warmlyremindingthemthateventheirexcrementwasadangertotheworld,bythenurseswearingPPE.Aftertheystoppedimprisoningsuchwomeninthe1950s,theprisonersremained.In1992,whentheasylumclosedforgood,thethreeremainingwomenweredeemedinsaneandrelocatedtootherinstitutions,theirentirelivesdestroyedbyaninfectiouspanic.Despitethis,theUKDepartmentofHealthtoldtheBBCthatthereneverhadbeenapolicyofincarceratingpeopledeemedcarriersofaninfectiousdisease[32].ThisdocumentisbasedonanexaminationofallantibodytestdocumentationsubmittedtotheUSFDA(FoodandDrugAdministration)andaseriesofantibodysurveysofgroupsofpeoplefromaroundtheworld.

A Little Background

COVID-19isallegedtobeanRNAvirus,sotheRNAwillbeinyourbodyassoonasyouareinfected.RT-PCRisanultra-sensitivetest(capableofreliablydetectingasfewasfivemoleculesofRNAinasample,andpossiblytriggeringonjustone)andthereforeshouldbepositivealmostimmediatelyafterinfection.1IgMantibodiesarebelievedtobeproducedbythebodyasgenericinfectionfighters,soonafterinfection.AninfectedpersonwillnotbeIgMpositiveimmediately,butwithinafewdaysatmost.Theseantibodiespersistforawhileaftertheinfectionisresolved,butthenfadeaway.

IgGantibodiesarebelievedtobeproducedbythebodyasveryspecificfightersofaparticularinvader,suchasCOVID-19.SomescientistsbelievertheytakelongerthanIgMtobeproduced,butallagreethattheypersistlongaftertheinfectionisresolved,possiblyforalifetime.

Antibodies and Antigens

Antibodiesarebelievedtobegeneratedbytheimmunesysteminresponsetoaforeignprotein,knownasanantigen.InthecaseofCOVID-19,anantigenwouldbeaproteinprobablyfoundontheoutershellofthevirus(becausetheinternalproteins

1Oftenonlysamplesfromsomeareasofthebodyarepositive(e.g.nosebutnotthroatorstool),leadingtothebeliefthatthevirus,unlikebloodborneviruses,onlycolonizesasmallpartoftherespiratorytract.Samplesfromdeepinthenose(nasopharyngeal)arebelievedtobemostreliableforearlydetection[27].

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areunlikelytostimulateanimmunereaction).Whenanantibodybindstoanantigen,itisasignaltothebodytodestroytheforeignobject,suchasavirusparticle.Antibodytestscontainoneofmoreoftheseantigens,thatareboundtochemicalsthatproducesomekindofcolorchangeorfluorescencewhenanantibodybindstothem.Theresultoftheantibodytestisreadastheintensityofthiscolorchangeorfluorescence.Thismakesreadingtestsresultseasiertoautomate.Theantibody-antigenreactioniscontinuous,andnotbinary,notnaturally‘negative’or‘positive’.Therefore,manufacturersrecommendaparticularintensityofcolorchangeorfluorescenceasthedivisionbetween‘negative’(antibodiesnotpresent)and‘positive’(antibodiespresent).Somemanufacturersrecommendanintermediatezonebetweennegativeandpositive,andspecimensinthiszonemaybere-tested,possiblyimmediately,orpossiblyinthefuture,whenitisbelievedthat,ifthereactionisreal,antibodylevelswillhaveincreasedtoaclearlydetectablelevel.Sinceantigensareviralproteinstheobviousplacetoobtainthemwouldbefrompurifiedvirus.However,sinceCOVID-19virushasneverbeenpurified,thisiscurrentlyimpossible.Inlieuofthis,traditional,impurematerials(e.g.nasalswab)wouldbeaddedtoacellculture,andproteinsthatwerebelievedtobeviralwouldbepurifiedandusedasantigens.Butinmoderntestsmostantigenproteinsare‘recombinant’,producedartificiallyfromthepublished30,000baseRNAsequencebelievedtobeCOVID-19.

Sources of Data

ThisarticleisbasedonareviewofallantibodytestsapprovedunderFDAEmergencyUseAuthorization[33],alistofsurveysmaintainedbyathirdparty[23]andseveralmedicalpapers.

Status of Antibody Tests

TheonlyjurisdictionwithaformalstructureforapprovalofantibodytestsistheUnitedStatesbut,untilveryrecently,itwasjustacharade,asthetestmanufacturersdidnotneedtoprovidevalidationdata.Now,validationdatamustbeprovided,buttheFDAcanonlydoapaperanalysis[3].Imagineifauto-manufacturershadtobuildcarstocertainEPA(USEnvironmentalProtectionAgency)fuelefficiencystandards,butratherthansendingacartotheEPAfortesting,theycoulddothetestingattheirfacilities,andjustsendtheresultsinafterwards.Then,therewouldhavebeennoneedtowritesoftwaretofakethefuelefficiencybyrunningtheenginedifferentlyundertestingconditions.

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A Theoretical Timeline

ThetheoreticaltimelineofanRNAvirusdiseaseisshownbelow:

Phase Description Exception

Pre-infection NoRNA,IgMorIgG Peoplewillhaveantibodiestopreviousinfectionsthatmaycross-reactwithCOVID-19antibodies.

Infection RNAshouldbedetectablebyRT-PCRalmostimmediately.

Incubation DuringthisperiodofafewdaysIgMantibodiesshouldbecomedetectable[1][2].IgGmaybecomedetectable.ItisbelievedthatIgGantibodiesdevelopatthesametimeorafterIgM,butnotbefore.

Symptomaticresolution

Ifapersondevelopssymptoms,theyshouldhavedetectableRNA,IgMandIgGduringthisperiod

AsymptomaticResolution

DespitethelackofsymptomsinmanyormostCOVID-19RNApositivepeople,peopleinthisphaseshouldsimilarlybepositiveforRNA,IgMandIgG.

Cure ThereisnofunctionalvirusleftinthebodysothepersonshouldbeRNAnegative.IgMandIgGwillbepositive.

RT-PCRtestsmayproducefalsepositivesresultsduetonon-infectiousRNAleftoverfromtheinfectionorotherreasons.

Post-infection AtsomepointIgMantibodieswaneandthepersonisleftwithjustIgGantibodies,whichprovideimmunity,possiblylife-long.

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AgraphfromthetestmanufacturerDiazymeillustratesthisbelief,whichindicatesthattheimmunesystemisawareoftheconceptofa7-dayweek(othersimilargraphsindicatethat,forotherviruses,multiplesof10daysarepreferred)[26].

ApaperfromtheJournaloftheAmericanMedicalAssociationdiffersinshowingIgMandIgGantibodiesarisingatthesametime[27]:

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ThispaperwillshowthatCOVID-19antibodytestingdoesnotsupportthistimeline.

Timeline in Practice

Pre-Infection: No Positive Test Results

BeforepeopleareinfectedwithCOVID-19theyshouldtheoreticallybenegativeforRNAandalltypesofantibodies.Inthefollowingtable,notethattestsforonlyoneantibodytypewillperformbetterastheyonlyhaveonechanceforafalsepositive,whereastestsformultipleantibodytypescouldtestpositiveforanytype.Thelowestvalueisshadedinblue,andthehighestinred.

Test or Study Antibodies Pre-outbreak samples positive2 (%)

Abbotttest[6] IgG 4/997(0.4%)

Cellextest[4] IgGorIgM 11/250(4.4%)

Chembiotest[5] IgGorIgM 5/125(3.6%)

DiaSorintest[9] IgG 8/1090(0.7%)

EuroImmuntest[10] IgG 30/1415(2.1%)

Idahosurvey[21] IgG 1/1020(0.1%)

Netherlandssurvey[24] IgA,IgG,IgM 30/218(13.8%)

Netherlandssurvey[24] IgM 3/28(10.7%)

Plateliaserum[8] Combined 3/687(0.4%)

Rochetest[13] Ngene 10/5272(0.2%)

Wadsworthtest[11] IgA,IgG,IgM 1/256(0.4%)

Infection: RNA-Positive Only

Theoretically,someonewhohasjustbeeninfectedwithCOVID-19willberapidlypositiveforRNA(duetothesensitivityofthetest)butitwilltakeafewdaysforantibodiestodevelop.Thereisnodataavailableatpresent,asitwouldrequiredailybloodsamplesfromalargenumberofpeoplewhowereinitiallynegativeforalltests,sothatthetimeseriescouldbeexamined.Thistypeoftestingcouldvalidateallaspectsofthetheoreticaltimeline,butwouldalsobeveryexpensive,intrusive(dailyswabsandbloodtests)andwouldneedaverylargenumberofpeople

2Sometestshada‘borderline’or‘indeterminate’categoryandthesewerecountedaspositive.

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becausemostmayneverhaveanypositivetestsand,aheadoftime,itwouldbeimpossibletotellwhowouldeventuallybecomeRNA-positive.ThebestthatcanbedoneistoguessatthedateofinfectionbasedoncontactwithsomeonewholatertestedRNApositive,butthereisneverproofthatthiswasactuallythedateofinfection,itisstilljustasupposition.

ThetimeatwhichsomeonefirstdevelopssymptomsorlearnstheyareRNA-positiveisnotveryusefulbecauseitmayoccuravariablenumberofdaysaftertheyareinfected.

Incubation: Antibodies Start to Develop

Thispartofthetheoreticaltimelinehasthesameproblemasthemomentofinfectionandwouldalsorequiretimeserieswithdailytestingofalargenumberofpeople.ButperhapswecansometimesbeluckyandsomeonewillbetestedearlyenoughthattheywillbeRNApositiveandthedevelopmentoffirstIgMandthenIgGantibodies.IntheChembiotestvalidationIgGantibodieswerefoundinallfourRNApositivesamplescollectedwithin6daysofthedevelopmentofsymptoms,butIgMantibodiesonlyinoneoutoffour[5].ItshouldhavebeentheotherwayroundifIgMoccursbeforeIgG.Astudyof30severelyandmildlyillCOVID-19patientsfoundthat,“ahigherproportionofpatients…hadearlierIgGthanIgMseroconversion[firstdetectionofantibodies]”[28].Sometestsandstudiesmadeitimpossibletovalidatethistheorybecausetheyusedtotalantibodies,notdistinguishingbetweenIgMandIgG(Platelia[8]).ManyothertestsonlyreactedtoIgGantibodies,socomparisonwithIgMwasnotpossible.ThereislimitedinformationbutitdoesnotsupportthenotionheldbysomethatIgMantibodiesdevelopbeforeIgG.ThisisconsistentwiththefirstSARScoronavirusinwhichIgGantibodieswerefoundbeforeIgMantibodies,callingintoquestiontheusefulnessofIgMantibodiesasanearlywarningsystem[25].And,giventhatIgMantibodiesdisappearovertime,theyarenotusefulfordetermininglaterimmunityeither.

Symptomatic Resolution: RNA and Antibodies

Oncesymptomsarenoticed,enoughtimeshouldhavepassedforIgMantibodiestodevelop,soduringthedaysorweeksofresolutionofsymptomseverypatientshouldbepositiveforRNA,IgMandIgG.DuringSARS,alsoblamedonacoronavirus,asmallsampleofisolatedpatientsmostlydevelopedIgGantibodiesby14daysaftersymptoms,andallby30days[25].

TheChembiotestfoundIgGantibodiesin100%ofRNA-positivesamplesfrom0-21daysafterfirstsymptoms,exceptfor4/10(40%)ofsamplescollectedbetween7-10

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days.TheEuroImmuntesthadpositiveIgGresultssporadicallyfromthefirstdayofsymptomsthroughday15,andthenconsistentlythroughday36,thelastdaytested,whilenegativetestswerefoundfromthedayofsymptomsthroughday18.Therewereverysmallnumbersoftestsperformedoneachday(1-6)withanaverageoflessthantwotestsperpatient[10].

TheAbbottIgGtesthad0positiveresultswithin3daysoffirstsymptoms,25%positivewithin3-7days,86%within8-13daysand100%after14days[6].Similarly,Diasorinfound11/44(25%)positiveforIgGwithin5daysoffirstsymptoms,44/49(90%)between6and14days,and40/41(98)after15days[9].TheOrthoVitriostestfound8%IgGnegativetothe‘N’genewithin5daysofthepersontestingRNApositive,butthefractionthenwentup,11%ontests6-15daysafterRNApositivity,and25%duringthe16-22dayperiod[12].Theyalsotestedpeopleaknownnumberofdaysaftersymptom,andagainasignificantfractionwerenegative:8%12-17daysaftersymptomsandmore,17%,18-32daysaftersymptoms.TheWadsworthtest[11]simultaneouslydetectsIgA,IgGandIgMantibodies,socannotbeusedtodistinguishthetimingofdifferentantibodies.However,itsurprisinglyhadnegativeresultson40%ofsamplesfrompeoplewhowereknowntohavebeenRNApositivefor11-15days,43%positivefor16-20days,and12%positiveformorethan20days.Ifindeterminateresultsareincludedwithnegative(sincetheyarenotclearlypositive)thepercentagesare69%(11-15days),51%(16-20days)and21%(over20days).TwoadditionalstudieswiththeWadsworthtestshowedthat,atleast25daysaftersymptomoffset,6%wereantibodynegative,and12%wereeitherantibodynegativeorindeterminate.Inotherwords,negativeresultsforIgA,IgGandIgMwerefoundlongaftersomeantibodiesshouldhavedeveloped.

Differenttestsgiveverydifferentresults,fromChembio,positiveforIgGonalldaysaftersymptomsdeveloped,toAbbottwhichhadonly25%positivewithin3daysofsymptomsdeveloping.Thisindicatesthatthetestsarenotallmeasuringthesamething,ornotwiththesamelevelofsensitivity.Additionally,therelevanttimingisfromthedateofinfection,notsymptoms,andthatisunknowninalmosteverycase.Asurveyof85COVID-19patientsinWuhan,ChinafoundthatthemajorityofsampleshaddetectableIgMantibodiesfromthefirstdaymeasuredto30daysorbeyond,buttherewasnotimewhereallteststakenwerepositive(themaximumwas94%onday19aftersymptoms).IgGsamplestakenonday30orlaterwere100%positive,butonly14outof85patientsweretestedduringthisperiod.Priorto30daysallgroupsofsampleshadatleast9%negative,andsomeasmuchas60%.Alargeflawinallthevalidationsisthatthepeoplesampledatdifferenttimesarenotthesame,soindividualanomalies(suchasthedisappearanceofIgGantibodies,andthenreappearance)cannotbeseen.Again,atimeseriescouldprovideinformationthatshowsthatthedevelopmentofantibodiesfollowsapredictablepatterninindividuals.

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TheonlythingapproachingatimelineisfoundintheAbbotttestdocumentationwhichshowstwopeoplewhohadtwonegativeIgGtestsfollowedbyseveralpositivetests.Thetwopeople,however,seroconvertedatratherdifferenttimes.Onebetweendays6and7aftersymptomsandtheotherbetween10and11[6].Asusual,theamountoftimefrominfectiontothedevelopmentofantibodieswasunknown.SincetheAbbotttestisIgGonly,therewasnoinformationonIgM.Insummary,formuchofthetestdocumentation,therewereamixtureofpositiveandnegativeIgGandIgMtestresultsovermuchofthetimetested,andforsometests,rightuptotheendoftheperiod.Thiscouldbeduetolargevariationsinthedevelopmentofantibodiesineachperson,falseresultsfromcertaintestkits,orboth.

Asymptomatic Resolution: RNA and Antibody Positive

FromatestingperspectivetheasymptomaticresolutionofaninfectionshouldalsobeatimewhenpeoplearepositiveforRNA,IgMandIgG.Theproblemisthatthepersonaffectedisnotsick,andmuchlesslikelytobetested.Again,atimeseriesofmanypeoplecouldidentifyasymptomaticinfections,andcouldtestthehypothesisthatthesepeoplewouldfirstbecomeRNApositive,thenIgMpositive,thenIgGpositivebeforetheresolutionoftheinfection.TheinformationthatisavailableontheappearanceofantibodiesinasymptomaticpeoplewhoareRNA-positiveisabsentthedateofinfection,whichistheonlydatethatmatters.Therefore,thereisnousefulinformationonthistheoreticalphase.

Post-Infection: Disappearance of IgM Antibodies

IgMantibodiesshoulddisappearafterapersonhaseliminatedthevirus(becomingRNAnegative).

TheChembiotestvalidationobtainedsamplesfrom2peopleat21daysaftersymptoms,andbothwereIgMpositive[5].Similarly,asurveyof85patientsinWuhan,China,followedpatientsforover30daysanddidnotdocumentthedisappearanceofIgM[29].Additionally,thedisappearanceofIgMantibodiesimpliesthattheyappearedinthefirstcase,buteveninpeoplewhoareRNApositivewithsymptoms,therearestillsometimesnegativeIgMtests.ThisisoftenmaskedbyconsideringsomeonewhoisIgMORIgGpositive,tobeantibodypositive.ThedocumentationavailabledoesnotexcludethepossibilitythatsomepeoplenevergeneratedIgMantibodies.ThedataprovidedinthisarticledoesnotsupportthenotionthatIgMantibodieseventuallydisappear,butitmayjustbebecausethepatientswerenotfollowedlongenough.

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Performance Issues

Positive Results on Coronavirus Negative People

COVID-19antibodytestsshouldonlyveryrarelybepositiveonpeoplewhotestedRNAnegative(whowerelikelytestedmultipletimes,usingsamplesfromdifferentareasofthebody),eveniftheywerehospitalizedforsymptomsthatmighthaveseemed‘COVID-like’butactuallytestedRNAnegative.ThereisalwaysthepossibilitythatsomeofthesepeoplepreviouslyhadaCOVID-19infection,probablyasymptomatic(otherwisetheywouldlikelyhavebeentested),butinnoneofthesecaseswasthereanyevidenceforthis.

Test or Study Antibodies RNA negative, antibody positive (%)

Autobiotest[7] IgGorIgM 3/312(1.0%)

Chembiotest[5] IgGorIgM 4/41(10.3%)

Wadsworth[11] IgA,IgG,IgM 1/30(3%)

Wu[22].Hospitalpatients. IgG 39/380(10.3%)

Wu[22].Returningworkers. IgG 98/1021(9.6%)

Xiangetal[29] IgG 20/84(24%)

Xiangetal[29] IgM 21/84(25%)

Antibody Measurement Performance

Antibodiesaregenerallymeasuredbyacolorchangewhichcanbemonitoredbyreflectance,fluorescenceoropticaldensity.Thecolorchangeshoulddeepen,orthefluorescentglowshouldincrease,withthequantityofvirusinapredictable(preferablylinear)fashion.Inotherwords,ifthebloodisdiluted50%thenthereflectance,fluorescenceoropticaldensityshoulddropbyhalf.

IntheChembiovalidation,whenbloodsampleswerecontinuouslydilutedbyhalf,theydidnotfollowapatternofopticalreflectancethatwasrelatedtotheamountofdilution.Withonesample,afterreflectancedroppedfrom36to16onthefirstdilution(closetohalf,asexpected)thereflectancestayedbetween11and16untilthefifthdilutionwhereitroseto24,whichwasalmostconsideredapositiveresult(25wasthecutoff).Onthesecondsample,theIgMreflectancealmostdoubledonthefirstdilution(asopposedtodropping).Thiswastheonlytestvalidationthatincludedasimilarexperiment,sothereisnoevidencethatantibodytestingresultscanbeusedtoestimatethequantityofvirus.Italsocallsintoquestionthemeaningfulnessofanumericcutoffinthefirstplace,todistinguishpositivefromnegative(andpossiblyborderlineorindeterminate).

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Disease Severity Predictive Value

Theamountofantibody,measuredbysurrogateslikereflectanceoropticaldensity,isoftenmeasured,withtheimplicationthatthelevelofantibodiesreflectstheseverityofthedisease.OnesurveyofCOVID-19patientsexaminedtwotypesofIgMandIgGlevels(anti-NP[internalnucleoprotein]andanti-RBD[surfacespikeproteinreceptorbindingdomain])foragroupof7severelyillpatientsandagroupofmildcaseandconcludedthat,“Serumantibodylevelswerenotcorrelatedwithdiseaseseverity”[28].Therewassimilarlynoobviouspatterninthesamestudywithpatientswithorwithoutco-morbidities.

ApaperfromShanghaistudiedantibodytiters(levels)in175recoveringCOVID-19patients,andfoundaweakcorrelationwithage,butnocorrelationwithpeoplewhoneverdevelopedhighlevelsofantibodiesandthedurationofdisease[31].

Cross Reactions

Antibodytestsareoftensubjecttocross-reactionswithotherconditions.Thiscouldbebecausethemedicalconditionproducessimilarantibodies,orbecausesomethingrelatedtothatconditionreactswithothertestcomponents.

Condition Test or Study Antibodies Positive (%)

ANA(anti-nuclearantibodies)

Euroimmun[10] IgG 1/29(3.4%)

Auto-antibodies Euroimmun[10] IgG 1/10(10%)

Chikungunyavirus Wadsworth[11] IgA,IgG,IgM 2/5(40%)

Chlamydophila EuroImmun[10] IgG 1/15(7%)

Coronavirus229E Chembio[5] IgG 1/1(100%)

Cytomegalovirus(CMV) Abbott[6] IgG 1/5(20%)

HepatitisBpositive Diasorin[9] IgG 1/10(10%)

HIV Wadsworth[11] IgA,IgG,IgM 1/5(20%)

InfluenzaApositive Diasorin[9] IgG 1/10(10%)

Mycoplasma EuroImmun[10] IgG 1/15(7%)

RespiratorySynctitiaVirus(RSV)

EuroImmun[10] IgG 1/3(33%)

Rheumatoidfactor Diasorin[9] IgG 1/10(10%)

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Streptococcus EuroImmun[10] IgG 2/12(17%)

WestNilevirus Wadsworth[11] IgA,IgG,IgM 1/5(20%)

Thechoiceofconditionstocheckforiscompletelyunderthecontrolofthemanufacturerandevenwhennocrossreactionswerefoundforacondition,thenumberofsamplestestedwassosmallthatthepossibilityofafairlyhighrateoffalsepositivecrossreactionsstillexists.Forexample,asampleof10cannotshowthatevena10%falsepositiverateisunlikely.

General Criticisms of Tests

Evenwheretestvalidationdataconformstotheexpectationaboutthebehaviorofantibodies,therearecriticismsthatcanbemade:

• Manufacturersareresponsibleforprovidingthedata,andtheyknowthereisnopointinsubmittingdatawithmajorredflags,meaningthattheycanspendtimeadjustingthesamplestheyareusing,andhowtheyareanalyzedtoensurethatthesubmittedreportmakestheirtestlooksgood.

• Thereisnowaytovalidatethemanufacturervalidationdata.• Thereisnoconsistentsetofvalidationteststhatneedtobeperformedbyall

manufacturers.• TimeseriesfromthetimeofinfectionthroughatleastthedeclineofIgM

antibodiesarenotprovidedinanycase.• Wheninformationisprovidedovertime,itisnotforthesamepeople.• Timingofantibodyresultsisfromthedayoffirstsymptoms,orthedayof

testingRNA-positive,notfromtheearlierdateofinfection.• Inmanyvalidationtestsonlytinynumbersofsamplesaretested.Sometimes

across-reactionwassearchedforbytestingonlyonesample.Yet,witheven1%crossreactionsbeingimportant,wellover100sampleswouldbeneeded.

• Onlyalimitednumberofconditionsweresearchedforcross-reactions.• Sincethetestswerevalidatedbythemanufacturersinidealenvironments,it

canbepredictedthatperformancewillbelowerwhenusedinpracticebypurchasersofthetests.

Theseflawsinantibodytestsarefatal.Atpresentnoantibodytestsareproperlyvalidated,andtheresultscannotbereliedupon,particularlynottomakesweepingchangesinsociety,suchasmandatoryvaccinationandquarantineofpeoplewhodonothavethe‘right’antibodytestresults.

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Population Surveys

Severalsurveysoflocalpopulationsforantibodieshavebeenundertaken.InmanycasesthisistoestimatethepenetrationofCOVID-19intothegeneralpopulation,whohavemostlybeenasymptomatic,orexperiencedonlyminorsymptoms.

Population being Surveyed

Itisveryhardtocomparethesesurveysbecausetheyusecompletelydifferentsamplesofpeople.Somearerandomhouseholdsurveys,althoughrandomizationmaybereducedbyallowingmultiplehouseholdresidentstoparticipate.Othersaresurveysofblooddonors,peoplewhohavegivenbloodatalabforreasonsunrelatedtoCOVID-19,volunteersrecruitedbyfacebookads,oratatestingcenterinapublicplace.Nosurveycanbetakenasrepresentativeofthegeneralpopulation.

Validating the Fraction Positive

Theresultofapopulationsurveythateveryoneisinterestedinisthepercentagepositive.Thisisgenerallymuchhigherthanexpectedbythosewhofocusonthenumberofknowncases,bydramaticallyexpandingthenumberoflikelycases.ThesesurveysleadtotheconclusionthatthedeathratefromCOVID-19isgreatlyexaggerated(especiallyintwoCaliforniasurveys)andthatherdimmunitymayoccurnaturally.Butthereisnoevidencethatthefractionsofthepopulationthatareantibodypositivearemeaningful,forseveralreasons:

• ThepresenceofantibodiesistakentomeanthatthepersonwaspreviouslyRNApositivewithnosymptoms,orminorsymptoms.Noneofthesurveyshaveproofthatallthepeople,orevenamajority,werepreviouslyRNA-positive(andpresumedinfected),andthetimehasobviouslypassedtoobtainthisinformation.

• ThepeoplewereassumedtobeantibodynegativepriortobecomingRNApositive.Noneofthesurveyshaveevidenceforthis.

• TheabsenceofantibodiesistakentomeanthatthepersonwasneverCOVID-19RNApositive.Noneofthesurveyshaveevidenceforthis.

• Itisassumedthatthetestsusedwouldallgiveapproximatelythesameresult.Sincetherehasbeennocross-validationoftests,thisisanunfoundedassumption.

Viruspurificationcannotbeusedtovalidateantibodytestswhenthevirusisbelievedtohavebeendefeatedandisnolongerinthebody.OnlyatimeseriescouldidentifypeoplewhobecomeRNA-positive,andthenmonitortheirantibodydevelopmentovertime.

Summary of Fraction Positive

ThissectioncontainsinformationfromantibodysurveysinatablemaintainedbyDeanBealer[23].Itshowsthegroupbeingsurveyed(cohort),thetypeofantibodies

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lookedfor(notalwaysprovided),thepercentagewhowereantibodypositiveand,insomecases,thepercentagewhowereasymptomaticintheweeksbeforethetest.Acronymsusedinthetable:HCW=HealthCareWorkers;HS=HighSchool;n/s=Notspecified;THL=in-houseantibodytest.

Region Cohort Antibodies Positive% Asymptomatic

Boston (Chelsea)

RNA negative volunteers n/s 32.0% 50.0%

Czech Selected or random n/s 0.4%

Denmark Blood donors 17-69 IgM 0.7%

Denmark Blood donors 17-69 IgG 0.7%

Denmark Blood donors 17-69 IgM+IgG 0.4%

Geneva Representative sample IgG 5.5%

Germany 405 households from random sample of 600

n/s 15.0%

Helsinki Gave blood sample at lab. Week 13

Rapid+THL 0.7%

Helsinki Gave blood sample at lab. Week 14

Rapid+THL 0.0%

Helsinki Gave blood sample at lab. Week 15

Rapid+THL 2.7%

Idaho General IgG 1.8%

Iran 196 households IgM or IgG 22.2% 55.6%

Kobe Outpatient blood tests IgG 3.3%

Los Angeles Representative sample n/s 4.1%

Madrid HCW IgA, IgG, IgM 9.3%

Miami-Dade Random county residents n/s 6.0% 50.0%

Moscow First week of survey n/s 3.0%

Moscow Second week of survey n/s 9-10%

Netherlands RNA negative blood plasma donors

IgA, IgG, IgM 3.1%

New York Recruits at grocery stores and community centers

n/s 12.3%

Oise, France HS pupils, staff IgG, anti-N gene 25.9% 17.0%

Oise, France Blood donors IgG, anti-N gene 3.0%

Santa Clara Facebook ads targeted at Santa Clara County

IgG or IgM 1.5%

Scotland Blood donors, March 17 n/s 0.0%

Scotland Blood donors, March 21-23 n/s 1.2%

Slovenia General n/s 3.0% 95.1%

Stockholm General n/s 2.3%

Sweden HCW n/s 20.0%

Switzerland Population representative survey, week 1

IgG 3.2%

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Switzerland Population representative survey, week 2

IgG 5.3%

Switzerland Population representative survey, week 3

IgG 8.7%

Switzerland Population representative survey, 5-19 years old

IgG 6.1%

Switzerland Population representative survey, 20-49

IgG 8.4%

Switzerland Population representative survey, 50+

IgG 4.3%

Switzerland Population representative survey, Female

IgG 3.2%

Switzerland Population representative survey, Male

IgG 5.3%

Wuhan RNA-negative returning workers IgG 9.6%

Wuhan RNA-negative returning workers IgM 0.0%

Wuhan RNA-negative hospitalized patients

IgG 10.3%

Wuhan RNA-negative returning workers IgM 0.0%

Atpresentthisinformationissimplyprovidedasaconvenientsummary.Drawingconclusionsfromitisdifficult,excepttosaythatiftheantibodytestscanbebelieved,innoareahavethemajorityofpeoplebeeninfected.Ontheotherhand,theresultsmaynotevenbeclosetothenumberofpeoplewhoactuallydidexperienceaninfectionasthereisnowaytovalidateanantibodytestinthegeneralpopulation,withouthistoricalrecordsofcoronavirus‘infection’status(i.e.atimeseriesdocumentingRT-PCRRNApositivityandthesubsequentdevelopmentofantibodies).Wherethefractionofpeoplewhohadbeenasymptomaticintheweeksbeforetheantibodytest(notonthedateofthetest,astheinfectionhaspresumablybeenresolvedsometimeago),amongagroupofantibodypositivepeople,wasreported,thenumberswerealloverhalf,exceptforthestudyinOise,France,inwhichparticipantswereaskedtoreportanyrespiratorysymptomsoverthelastthreemonths,whichweremostlyrunnynose,cough,headache,tiredness,sorethroatandfever.Abouthalfwerelistedashaving“major”symptoms(sohalfwereasymptomaticorhadminorsymptoms),but‘major’symptomsincludedfever,coughandlossofsensationsofsmellortaste.Thebottomlineisthatifwedefinemajorsymptomsbytheneedforhospitalization,95%didnothavemajorsymptoms.

Antibodies and Air Pollution

AnantibodysurveyofNewYorkprovideddataforvariousregionsofthestate.ThereisanobviouslyhigherrateofantibodypositivepeopleintheNewYorkCityarea,andadramaticallylowerrateinruralareas.Thiscouldbeexplained(andwillbe)bygreatertransmissioninthecity,butalsocouldbeduetogreaterairpollutioninthecity.Therearealreadystudiesthatshow,forexample,anassociationbetweenairpollutionandthefrequencyofRNApositivetests,andbetweenairpollutionanddeathsblamedonCOVID-19.

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Onestudyestimates,“Anincreaseof1microgrampercubicmeteroffineparticulatesintheairisassociatedwithan8%increaseintheCOVID-19deathrateintheUnitedStates”[16].AnotherstudyfoundasimilarcorrelationinChina,ItalyandtheUSAusingsatellitemeasuresofparticulatematter,CarbonMonoxideandNitrogenDioxide[18].AstudyinEnglandcorrelatedCOVID-19lethalitywithNitrogenOxide,NitrogenDioxideandOzonelevels[19].AnItalianstudyshowedaveryhighcorrelationbetweenthenumberoftimesparticulatematterlimitswereexceededinanareaandthenumberofinfected(i.e.RNA-positive)people.Mostofthepollutedareas,bythismeasure,wereinnorthernItaly[17].AstudyinLondon,EnglandshowedastrongcorrelationbetweenhigherairpollutionandhighernumbersofRT-PCRRNAtestrates[20].ReturningtotheNewYorkdata,thehighestfractionofpeoplewhotestedantibodypositiveaftervolunteeringfortestingatgrocerystoresandcommunitycenterswasinNewYorkCity(20%)followedbyWestchester/Rockland(14%)andLongIsland(11%).TheregionswiththelowestfractiontestingpositivewereSouthernTier(2.4%),CapitalDistrict(2.2%)andCentralNY(1.9%).SouthernTierisahillyandagriculturalareaonthesouthernborderofthestate.TheCapitalDistrictcontainsthecityofAlbany,andisdependentlargelyongovernment,healthcareandeducationemployment.CentralNYcontainsthecityofSyracuse.Whileonceindustrial,mostemploymentisnowineducation,research,healthcareandservices.Thisevidenceisfarfromproofthatfalsepositiveantibodytestscanbeinducedbyhighlevelsofairpollution,butgiventhatRNApositivityandCOVIDdeathsareassociatedwithairpollution,itisahypothesisthatshouldbeconsidered.

Review of Timeline

Basedonthefindingsinthispaperwecanreviewtheevidenceforthetheoreticaltimeline.

Phase Description Exception

Pre-infection NoRNA,IgMorIgG. Asignificantnumberofoldbloodsampleshadantibodiesdetected.

Infection RNAshouldbedetectable. Outsidethescopeofthispaper.

Incubation IgMantibodiesshouldbecomedetectable[1][2].IgGmaybecomedetectable.

LimitedinformationasinfectionisusuallyonlydeclaredoncesymptomsorapositiveRNAtestisfound.Thedateofinfectionisneverknown.

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Symptomaticresolution

Ifapersondevelopssymptoms,theyshouldhavedetectableRNA,IgMandIgGduringthisperiod

Thereareseveralcaseswheresamplesarenegativeforallantibodiestested.

AsymptomaticResolution

DespitethelackofsymptomsinmanyormostCOVID-19RNApositivepeople,peopleinthisphaseshouldsimilarlybepositiveforRNA,IgMandIgG.

ThereislimitedinformationsincemostasymptomaticpeoplearenottestedforRNA.

Cure ThereisnofunctionalvirusleftinthebodysothepersonshouldbeRNAnegative.IgMandIgGwillbepositive.

AsignificantnumberofsamplesarenegativeforIgMandIgGevenmanydaysafterfirstsymptoms.

Post-infection AtsomepointIgMantibodieswaneandthepersonisleftwithjustIgGantibodies.

AsignificantnumberofsamplesarenegativeforIgG.Thereisnoinformationonimmunityfromre-infection.

Conclusions

PositiveCOVID-19antibodytestshaveonlybeenfoundinaminorityofpeopleinthegeneralpopulationevenwherethevirusisbelievedtohavebeencirculatingformonths.Thesefractionsaregenerallytakenastruth,butonewouldexpectahighlyinfectiousvirustohavespreadmuchmorewidely.Thereisalotridingonthisdata,ifonlyasmallminorityofpeoplehaveCOVID-19IgGantibodies,thenitmaybedeclaredbyvaccineproponentsthatnaturalimmunityisnotpossible,andthatavaccinemaystillbenecessary,evenmandatory.

Thefaithinthisdataishardtounderstandsincethereisnoevidencethatthevastmajorityofpeopleinsurveyswereever‘infected’(i.e.wereeverRNApositive)andnoevidencethattheantibodiesseenduringthesurveywerenotpresentinthepast.Ontheotherhand,thereisalsonoevidencethatthemajoritywhotestnegativeweretrulynever‘infected’(i.e.neverwereRNApositive).

Determiningimmunityisalsovirtuallyimpossible.Obviouslytherewouldbeethicalproblemsre-challengingpeoplewithavirusthatisbelievedtobefatalinsomepeople.Thereare,however,asignificantnumberofpeoplewhotestRNApositiveaftersymptomshaveresolved,andaftertestingRNA-negative.Thiscouldbeusedasevidencethatre-infectionispossible(strengtheningthecaseforavaccine)butgiventhatthesepeopleareasymptomatic,mayjustindicatefalsepositives[30].

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ThereisnoevidencecurrentlythatthepresenceofIgGantibodiespreventspeoplefrombecomingRNApositiveagainor,conversely,thattheabsenceofIgGantibodiesmakespeoplevulnerabletobecomingRNApositive.ProofthatagroupwithoutCOVID-19IgGantibodiesaremorevulnerablecouldnotjustlookatthere-occurrenceofRNA,becausethatusuallyoccurswithoutsymptoms.EvenifoccurrenceofRNAwithsymptomsismorecommon,onewouldhavetoshowthattheoverallriskofseriousillnessanddeathwashigher,afteradjustingforbaselinedifferencesbetweenthegroupswithandwithoutIgGantibodies.Theoneexperimentthatcouldshowwhetherantibodytestsareactuallymeaningfulwouldbeatimeseriesofalargenumberofpeoplewhoarecurrentlynegativeonalltests.Thisexperimentwouldbetimeconsuming,inefficient(asmanypeoplewouldneverbecomepositiveonanytests),intrusive(frequentnasalswabsandbloodtests)andobviouslyveryexpensive.Thosearepracticalconsiderations,butintheabsenceofsuchanexperimentwearealmosttotallyinthedarkaboutCOVID-19antibodytesting.GiventhebillionsbeingspentonCOVIDandthetrillionsbeinglostbytheeconomy,itsurelyisnotimpossibletodosomeworthwhilescience.Additionally,ifviruswaseverpurifiedfrompeoplewhowereRNApositiveandsymptomatic,thiscouldbeusedtoexposeanimals,andcouldbeusedtodetectantibodiesthataredefinitelyfromCOVID-19,andnotjusttoproteinsderivedfromtheputative30,000baseCOVID-19genome.

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Revision History Revision Major changes

3 Editorialrevisions.Executivesummary.Secondpapershowingnocorrelationbetweendiseasesymptomsandantibodylevels.