Molecular and immunological approaches in quantifying the air-borne food allergen tropomyosin in crab processing facilities Paper I

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Paper I

Molecular and immunological approaches in quantifying the air-borne

food allergen tropomyosin in crab processing facilities

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InternationalJournalofHygieneandEnvironmentalHealthxxx(2014)xxx–xxx

ContentslistsavailableatScienceDirect

International Journal of Hygiene and Environmental Health

j ourna l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / i j h e h

Molecular and immunological approaches in quantifying the air-borne food allergen tropomyosin in crab processing facilities

Sandip D. Kamath

^a,b

, Marte R. Thomassen

^c,e

, Shruti R. Saptarshi

^a,b

, Hong M.X. Nguyen

^d

, Lisbeth Aasmoe

^c,f

, Berit E. Bang

^c,f

, Andreas L. Lopata

^a,b,∗

aMolecularImmunologyGroup,SchoolofPharmacyandMolecularScience,JamesCookUniversity,Townsville,QLD,Australia

bCentreforBiodiscoveryandMolecularDevelopmentofTherapeutics,JamesCookUniversity,Townsville,QLD,Australia

cDepartmentofOccupational-andEnvironmentalMedicine,UniversityHospitalofNorthNorway,Tromsø,Norway

dFacultyofFoodScienceandTechnology,NongLamUniversity,HoChiMinhCity,VietNam

eDepartmentofCommunityMedicine,FacultyofHealthSciences,UniversityofTromsø,Tromsø,Norway

fMedicalPharmacologyandToxicology,DepartmentofMedicalBiology,FacultyofHealthSciences,UiTTheArcticUniversityofNorway,Tromsø,Norway

a r t i c l e i n f o

Articlehistory:

Received27January2014

Receivedinrevisedform8March2014 Accepted8March2014

Keywords:

Tropomyosin Allergendetection Occupationalallergy Shellﬁsh

SandwichELISA Airborneallergen

a b s t r a c t

Tropomyosinisacross-reactiveallergenicproteinpresentiningestedshellfishspecies.Exposureand sensitizationtothisproteinviainhalationisparticularlyimportantinthecrustaceanprocessingindustry whereworkersarecontinuouslyexposedtotheaerosolizedformofthisallergen.Theaimofthisstudy wastodevelopanantibody-basedimmunoassaytoenablethespecificandsensitivequantificationof aerosolizedtropomyosinpresentintheenvironmentoftwocrabprocessingfacilities.

Anti-tropomyosinantibodywasgeneratedinrabbitsagainsttropomyosinsfromfourdifferentcrus- taceanspecies.Theseantibodieswerepurifiedusingrecombinanttropomyosinusinganimmuno-affinity column.TherecombinanttropomyosinwasalsousedasanallergenstandardforthesandwichELISA.In ordertoquantifyaerosolizedtropomyosin,aircollectionwasperformedinthepersonalbreathingzoneof 80workersduringtwocrabprocessingactivities,ediblecrab(Cancerpagurus)andkingcrab(Paralithodes camtschaticus)usingpolytetrafluoroethylenefilters.Thepurifiedantibodywasabletodetecttropomyosin selectivelyfromdifferentcrustaceansbutnotfromvertebratesources.Thelimitofdetection(LOD)for thedevelopedsandwichELISAwas60picogram/m³andlimitofquantitation(LOQ)100picogram/m³. Immunoassayvalidationwasbasedonlinearity(R²0.999),matrixinterferencetest(78.8±6.5%),intra- assayCV(9.8%)andinter-assayCV(11%).Thenovelimmunoassaywasabletosuccessfullyidentify workingactivities,whichgeneratedlow,mediumorhighconcentrationsoftheaerosolizedfoodallergen.

Wedescribe an IgG antibody-based immunoassayforquantification of the majorfood allergen tropomyosin,withhighsensitivityandspecificity.Thismodifiedimmunologicalapproachcanbeadapted forthedetectionofotheraerosolizedfoodallergens,assistingintheidentificationofhigh-riskallergen exposureareasinthefoodindustry.

Abbreviations: Tm,tropomyosin;rTm,recombinanttropomyosin;cAb-␣TM, anti-tropomyosincaptureantibody;dAb-␣TM,detectionantibody.

∗Correspondingauthorat:MolecularImmunologyGroup,SchoolofPharmacyand MolecularScience,CentreforBiodiscoveryandMolecularDevelopmentofThera- peutics,Building21,MolecularSciences,JamesCookDrive,DouglasCampus,James CookUniversity,Townsville,QLD4811,Australia.Tel.:+610747814563;

fax:+610747816078.

E-mailaddresses:sandip.kamath@jcu.edu.au(S.D.Kamath),

marte.renate.thomassen@unn.no(M.R.Thomassen),Shruti.saptarshi@jcu.edu.au (S.R.Saptarshi),nmxuanhong@yahoo.com(H.M.X.Nguyen),

lisbeth.aasmoe@unn.no(L.Aasmoe),berit.bang@unn.no(B.E.Bang), andreas.lopata@jcu.edu.au(A.L.Lopata).

Introduction

Occupationalallergyandasthmahasbecomeaserioushealth concern,especiallyforworkersintheseafoodindustry.Increased globalconsumptionandchangingdietaryhabitshavegreatlyfacil- itatedseafoodproduction(LopataandJeebhay,2013;Lopataand Lehrer,2009).Thisinturn,hascausedmoreworkerstobeexposed toseafoodallergensonadailybasis.Accordingtoareportbythe FoodandAgricultureOrganization(FAO)in2010,nearly45mil- lionpeopleareinvolvedinseafoodandaquacultureproduction.

Severalstudieshaveshownthattheprevalenceofoccupational asthmaamong workersexposed toshellﬁsh isbetween4%and 36% (Bonlokke et al., 2012; Granslo et al., 2009; Howse et al., 2006).Moreover,workerswithoccupationalasthmatoshellﬁsh http://dx.doi.org/10.1016/j.ijheh.2014.03.006

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were shown to develop ingestion-related food allergies to the sameshellﬁshspecies(Jeebhayetal.,2001).Occupationalexposure toshellﬁshallergenscanelicitupperandlowerrespiratorytract symptomssuchasasthma,rhinitisandcanevencauseskinsymp- toms(Aasmoeetal.,2005;Bangetal.,2005;LopataandJeebhay, 2013).

In the seafood industry, workers are constantly exposed to air-borneshellﬁsh particulatematter arisingfrom thedifferent processingtechniques.Severalstudieshaveshownthepresenceof allergenicproteinsinair-borneparticulatematter.Theseallergens areresponsibleforcausing allergicsensitizationamongaffected workers(AbdelRahman etal., 2011,2013;Taylor etal., 2000).

SerumIgEantibodyreactivitytocrabproteinsamongsnowcrab processingworkersduetooccupationalexposureofcrabmatter hasbeenreportedpreviously(Cartieretal.,1984,1986;Gilletal., 2009; Malo et al., 1997; Weytjens et al., 1999).Aerosolization of shellﬁsh allergens occurs due to processes such as ﬁlleting, freezing, cooking, smoking, drying and techniques using high pressurewaterorair (Jeebhayetal.,2001; LopataandJeebhay, 2013). Processessuchas butchering, de-gillingand particularly boiling,havebeenshowntocauseexcessivebioaerosolformation.

Thewetordryair-borneparticlesmaythenbeinhaledbyexposed workers.AbdelRahmanetal.demonstratedelevatedlevelsofair- bornecraballergensinspeciﬁcworkstationssuchasbutchering andcookingascomparedtocleaning,packingandstorage(Abdel Rahmanetal.,2012).

The commonly consumed shellfish can be divided in two groups;crustaceans(shrimps,crabs,lobsters)andmollusks(oys- ter,mussels,octopus,squid) (Lopata and Kamath,2012; Lopata etal.,2010).Themajorshellfishallergenisa33–36kDamuscle protein called tropomyosin (TM). Over 80% of shellfish sensitized patients are known to react to this major allergen. As a muscle protein, tropomyosin exists in a dimeric confirma- tionwhich complexeswithtroponintocover theactin-binding sites during muscle contraction.Due tothis role, tropomyosin isa highly expressedprotein foundmainlyin theedible meat.

Tropomyosindisplaysaremarkablestabilitytoheatingandisable toretainitsallergenicity eveninheat-processed shellﬁshprod- ucts(Abramovitchetal.,2013;Johnstonetal.,2014;Kamathetal., 2013).

Tropomyosindisplayshighaminoacidsequence(primarystruc- ture) identity within crustaceans, ranging from 95% to 100%

(Kamathetal.,2013).Interestingly,crustaceantropomyosinshares acertaindegreeofaminoacidhomologyof75–83%sequenceiden- titywithhousedust-miteandinsecttropomyosins.Avarietyof miteandcockroachallergenshave beenimplicatedinair-borne exposureandsensitization,however,tropomyosinisonlyaminor allergeninbothallergenssources.Thisismostlikelyduetolow immunologicalcross-reactivityaswellaslowrelativeabundance ofthisproteincomparedtoconsumedshellﬁsh(Arlianetal.,2009;

Pomesetal.,2007).

Several studies have shown that antibody reactivity to tropomyosinisagoodpredictorofshellﬁshallergy(Gámezetal., 2011;Kamathetal.,2013).Duetoitsexcellentstructuralstability anddetailedcharacterization,tropomyosinwaschosenasanideal molecularmarkerfordetectingair-borneshellﬁshallergensinthis study.

Wereportthedevelopmentand validationofahighlysensi- tiveimmunoassaytodetectandquantifyaerosolizedtropomyosin inairsamplescollectedfromcrabprocessingfactories.Usingthis immunoassay,wewereabletoquantifyair-bornetropomyosinin aworker-andactivity-speciﬁcmanner.

Theapproachofusingarecombinantproteinasstandardand puriﬁed natural allergen to generate the capture antibody for increasedsensitivityandspeciﬁcityhasnotbeenemployedpre- viously.

Thismethodologycanbemodiﬁedforthequantiﬁcationofother majorfoodallergensandwouldbeanimportanttoolinmonitoring air-borneallergenlevelsindifferentworkenvironments.Thiscan subsequentlyassistinestablishingsafetyparadigmstocontrolthe unintentionalgenerationofaerosolizedallergensandaccidental sensitizationofexposedworkers.

Methods

Allergenstandard:expressionandpuriﬁcationofrecombinant tropomyosin

Recombinant tropomyosin was expressed and purified as describedpreviously(Kamathetal.,2013).Briefly,totalRNAwas extracted from fresh specimens of black tiger prawn (Penaeus monodon)usingtheRNAeasyminiextractionkit(Qiagen,Hilden, Germany)accordingtothemanufacturer’sinstructions.Comple- mentaryDNA(cDNA)wasreversetranscribedfromthetotalRNA usingtheTranscriptorHighFidelitycDNA SynthesisKit (Roche, Basel,Switzerland),followingthemanufacturer’sinstructions.The codingregionfor tropomyosinwasamplifiedbyPCR usingfor- ward5-GCGGATCCGACGCCATCAAGAAGAAGATGC-3andreverse 5-GCGAATTCTTAGTAGCCAGACAGTTCGCTG-3 primers. The PCR conditionsweresetasfollows,94^◦Cfor2min,30cyclesof94^◦Cfor 20s,55^◦Cfor20s,72^◦Cfor30sandafinalelongationstep,72^◦C for7min.The860bpamplifiedproductwasclonedintotheexpres- sionvectorpRSET-AusingtheBamH1andEcoR1restrictionsites.

Therecombinantexpressionvector,pRSET-A-TMwastransformed intoBL21Escherichiacolistrainandexpressionoftherecombinant tropomyosinwithaHIS-tag,inducedusing1mMIsopropyl␤-d- 1-thiogalactopyranoside(Amresco,USA).Thebacterialcellswere washedwithextractionbuffer(25mMTris–HCl,pH8.0,300mM NaCl,1mMimidazole)andlysedusingaFrenchpressurecell.After centrifugationat6000×gfor15min,therecombinanttropomyosin waspurifiedusing HIS-TrapFFAffinityColumn (GEHealthcare, USA).Thefractioncontainingtherecombinantproteinwasfurther purifiedusingaSuperdexG7516/600sizeexclusioncolumn(GE Healthcare,USA)onaBiologicDuoflowFPLC(BioRad,Hercules,CA, USA).Thepurifiedrecombinanttropomyosin waslabeled“rTm”

andstoredinaliquotsat−80^◦Cuntilfurtheruse.

Sodiumdodecylsulphate-polyacrylamidegelelectrophoresis (SDS-PAGE)

SDS-PAGEwasperformedasdescribedearlier(AbdelRahman etal.,2010)toconﬁrmthepurityofthetropomyosinstandardand analyzethebindingcharacteristicsofthepuriﬁedantibodies.

Twelve micrograms of the protein samples was heated in Laemmlibuffercontainingdithiothreitolandloadedontoa12%bis- acrylamidegel.Proteinseparationwasperformedat180Vusinga Mini-ProteanTetraCellelectrophoresissystem(BioRad,Hercules, CA,USA).Theseparatedproteinswerevisualizedbystainingwith CoomassiebrilliantblueR250(BioRad,Hercules,CA,USA).

CDspectroscopyofallergenstandard

Circulardichroismspectroscopywasperformedtoanalyzethe alpha helicalcontent of rTMand compare it topurified natural prawn tropomyosin. Natural and recombinant tropomyosin sampleswerepreparedinPBS,pH7.2andadjustedtoafinalcon- centration of 3␮M.CD spectroscopy wasperformed ona J715 Spectropolarimeter(Jasco,USA)withcontinuousnitrogenflushing at25^◦C.Allmeasurementswereperformedusinga10mmquartz cuvetteoverawavelengthrangeof190–260nm.Forwavelength analysis,thetropomyosinsampleswerescannedwithastepwidth of0.2nmandbandwidthof1nmat100nm/minaveragingover

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S.D.Kamathetal./InternationalJournalofHygieneandEnvironmentalHealthxxx(2014)xxx–xxx 3

eightscans.Finaldatawasexpressedasmeanresidualellipticity ()aftersubtractingPBSblankspectrum.

Productionofpolyclonalanti-tropomyosinantibodies Proteinextractionandestimation

Topreparetheantigenmixturetogenerateantibodiesinrabbits, protein extracts were generated from four crustaceanspecies;

Blacktigerprawn (Penaeusmonodon), Vannameiprawn(Litope- naeusvannamei),Bananaprawn(Fenneropenaeusmerguiensis)and School prawn (Metapenaeus macleayi) as described previously (Kamathetal.,2013).Thecompleteshellfishspecimen,initsouter shell,washeatedinliquid(PBS)at100^◦Cfor 20min.Theouter shellofthespecimenwasthenremovedandtheedibletissuecut intosmallpieces.Fiftygramsofthemusclemasswashomoge- nizedin150mLofphosphatebufferedsaline(PBS)for10minusing anUltraturraxblender(IKA,Staufen,Germany),agitatedfor3hat 4^◦Cfollowedbycentrifugationat14,000rpmfor15min.Thesuper- natantwasclarifiedthroughaglassfiberfilter,followedbyfiltration througha0.45␮mmembranefilter(Millipore,Billerica,MA,USA) andstoredat−80^◦Cuntilfurtheruse.

Tocharacterize thegeneratedpolyclonal antibody,unheated proteinextractswerepreparedfromprawn(Penaeusmonodon), crab(Portunuspelagicus),lobster(Jasusedwardsii),housedustmite (Dermatophagoidespteronyssinus),ﬁsh(Latescalcarifer),andpork (Susscrofa)asdescribedelsewhere(Abramovitchetal.,2013).

Tropomyosin–antigenmixpreparation

TheproteinextractsproducedinSection“Proteinextractionand estimation”wereusedasstartingmaterialtopurifytropomyosin.

TenmilligramsoftheproteinextractwasloadedonaminiMacro- prepHighQstronganionexchangecolumn,(BioRad,Hercules,CA, USA)in30mMTris–HCl,pH6.5.Tropomyosinwaselutedfromthe columnusingalineargradientwithincreasingsodiumchloridesalt concentrationinTris–HClbufferfrom0.4Mto0.6M,over20col- umnvolumes.ThepurifiedTMfractionswerepooledandabuffer exchangewithphosphatebufferedsaline(PBS)performedusing Amikonspinfilterswitha3kDamolecularweightcut-off(Milli- pore,Billerica,MA,USA).Thetropomyosinfractionsfromthefour crustaceanspeciesweremixedandadjustedtoafinalconcentra- tionof1mg/mLinsterilePBS.

Rabbitimmunization

TheimmunizationofrabbitswasperformedatInstituteofMedi- calandVeterinaryScience(IMVS),Adelaide,Australia.NewZealand rabbitswereinjectedwiththepreparedtropomyosinimmunogen alongwithFreund’sadjuvantinfourdosesattwo-weekintervals.

Thepre-bleedwascollectedatweek0toserveasanegativecontrol.

Atestbleedwascollectedatweek7totesttheproductionofthe antibodiesandtheﬁnalbleedconductedatweek9.Thecollected serumwasstoredat−80^◦Cuntilfurtherprocessing.

EnrichmentandpuriﬁcationofpolyclonalIgGanti-tropomyosin antibodies(captureantibodies)

ForthedevelopmentofthesandwichELISA,IgGantibodieswere enrichedfromtherabbitserumusingsodiumsulfateprecipitation.

Sodiumsulfate(Sigma–Aldrich,USA)wasaddedto10mLofserum insmallquantitiesatatime,toaﬁnalconcentrationof18%(w/v).

Theserumwasthencentrifugedat1500×gfor10minatambient temperature,thepelletresuspendedin18%(w/v)sodiumsulfate solution,re-centrifugedandﬁnallyre-dissolvedin5mLofsterile PBS,pH7.2.TheIgGenrichedserumfractionwassubsequentlydia- lyzedagainstPBSovernightandthenstoredat−80^◦Cuntilfurther use.

TheAminolinkPlusImmobilizationkit(ThermoScientiﬁc,Mel- bourne, Australia) was used to preparea tropomyosin afﬁnity

columnfortheisolationandpuriﬁcationoftropomyosinspeciﬁc IgGantibodiesfromtheIgGenrichedrabbitserumfraction.Thepro- cedurewasfollowedaccordingtothemanufacturer’sinstructions.

Briefly,rTmwascovalentlyboundtothecolumn,theremaining active sites blocked and washed, and equilibrated in PBS, pH 7.2.TheIgGenrichedrabbitserumfractionwasloadedontothe tropomyosinaffinitycolumnandincubatedfor30min.Theanti- tropomyosinIgGantibodyfractionwassubsequentlyelutedusing 0.2M glycine hydrochloride, pH 2.5 and neutralized with 1M Tris–HClpH8.5.Thepurifiedantibodyfractionwasthendialyzed againstPBS,pH7.2andstoredinaliquotswith0.05%sodiumazide at−20^◦Cforfurtheruse.ThisfractionwaslabeledcAb-␣TM.

Biotinylationofdetectionantibodies

Afractionoftheanti-tropomyosinIgGantibodieswerebiotiny- lated using the EZ-Link^® Sulfo-NHS-LC-Biotin (Thermo Fischer Scientiﬁc, Melbourne, Australia) following the manufacturer’s instructions.A100foldmolarexcessofbiotinwasusedtobiotiny- late theantibodiesfroma freshly prepared10mMbiotinstock solution.Afterbiotinylation,theantibodysolutionwasdialyzed againstPBS,pH7.4toremovetheexcessreactants.Sodiumazide wasaddedtotheantibodysolutiontoaﬁnalconcentrationof0.05%

(w/v)andstoredat4^◦CinambercoloredtubesandlabeleddAb-

␣TM.

Immunoblotting

SerumIgEimmunoblottingwasperformedtotestpatientIgE reactivitytorTmasdescribedpreviously(Abramovitchetal.,2013;

Johnstonetal.,2014).Serawerepooledfromfoursubjectswitha convincingclinicalhistoryofallergicreactivitytoingestedshellfish andconfirmedIgEbindingtonaturalpurifiedprawntropomyosin.

ThesubjectswererecruitedbyTheAlfredHospital,AllergyClinic, Melbourne,Victoria,Australia.

Toanalyzethebindingcharacteristicsofthecaptureantibody tovariousantigenicsources, theseparated proteinsonanSDS- PAGEgelweretransferredtoapolyvinylidenediﬂuoride(PVDF) membraneusingtheSemi-dryTransBlotApparatus(BioRad,Her- cules,CA,USA).Afterblockingwith1%(w/v)bovineserumalbumin (BSA)inPBS-T,themembranewassubsequentlyincubatedwith the capture antibody, cAb-␣TM, diluted 1:10,000 in 0.5% BSA, PBS-T and goat anti-rabbit IgG antibody conjugated with HRP (Promega,USA)diluted1:40,000.Themembranewasvisualized using the enhanced chemiluminescent technique as previously reported(AbdelRahmanetal.,2011).

Tropomyosinaminoacidsequencealignment

To predict the capture antibody binding characteristics to shellﬁsh tropomyosin, a protein sequence alignment was performed to compare vertebrate and invertebrate tropomyosin.

cDNA based protein sequences for tropomyosin were collected fromtheNCBI databasewiththefollowingaccession numbers;

Blacktigerprawn,accessionnumber (AAX37288.1),Blue swim- mer crab(AGE44125.1), Rocklobster(AFY98827.1), Housedust mite (ACI32128.1), German cockroach (AAF72534.1), Cod ﬁsh (BAC44994.1) andPigtropomyosin (NP001090952.1).Sequence alignment,matrixidentityscoresandpercentsimilaritywerecal- culatedusingClustalOalgorithminJalviewprogram(Waterhouse etal.,2009).

TheevolutionaryhistorywasinferredusingtheMinimumEvo- lution method.Thetreeis drawnto scale,withbranchlengths inthesameunitsasthoseoftheevolutionarydistancesusedto inferthephylogenetictree.Theevolutionarydistanceswerecom- putedusingthePoissoncorrectionmethodandareintheunitsof thenumberofaminoacidsubstitutionspersite.TheMEtreewas

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searchedusingtheClose-Neighbor-Interchange(CNI)algorithmat asearchlevelof1.Theanalysisinvolved7aminoacidsequences.

Therewereatotalof284positionsintheﬁnaldataset.Evolutionary analyseswereconductedinMEGA6.

Airsamplingandelutionofaerosolizedallergen,tropomyosin Collection of aerosolized tropomyosin was performed using polytetrafluoroethylene(PTFE)filterswithaporesizeof1.0␮m (Millipore, Billerica, USA). The filter cassette apparatus was attachedtoapump(SKCLtd.,UK)throughatubeandtheairflow adjustedto2.5L/min.Theaverageairflowratethroughthefilter, wascalculatedasthemeanofinitialandfinalairflowatthestart andstopofthepump,respectively.Whereachangeintheairflow fromstarttofinishwasmorethan10%,thesampleswerediscarded.

Theair-collectionapparatuswassetupinabackpackwhichwas carriedbyeachworkerduringtheirnormalshift.Theaircollection inletwasplacedintheworkerspersonalbreathingzone(PBZ)so astosampletheairavailableforbreathing.

ElutionofthecollectedallergenswasperformedusingNunc- ImmunoMinisorp tubes (Nunc, USA) to minimize the loss of allergencontentdue toadsorptiononthetubewalls.The PTFE ﬁlterswereremovedfromthecassettesandplacedintubescon- taining1mLofPhosphateBufferedSaline(PBS)with0.5%Tween 20and0.2mMSodiumazide.Theextractionwasperformed,by placingthetubesonarotationtilterfor2hatroomtemperature.

Theeluate wastransferredtoa newMinisorptubeand Bovine SerumAlbumin(BSA)addedtoaﬁnalconcentrationof1mg/mL.

Thiseluatewasthenaliquotedin200␮Lvolumeandstoredinmini Eppendorftubesat−80^◦Cuntilfurtheranalysis.Acleanunused ﬁlterwaseluted simultaneouslyusingtheextractionprocedure aboveandregardedasablanksample.

Assayprocedure

Allincubationswereperformedwith100␮L/wellatroomtem- peraturefor1hunlessotherwisestated.Allwashingstepswere performedusingPBSwith0.05%(v/v)Tween20,pH7.2(PBS-T) andrepeatedthreetimesonanEL405Autoplatewasher(BioTek Instruments,Winooski, USA)unlessotherwise stated.A96well highbinding Costar microtitre plate (Sigma–Aldrich, USA) was coatedwithcAb-␣TMdiluted1:500incarbonatebuffer, pH9.6 and incubatedovernightat 4^◦C. Afterwashing, thewells were blockedwith270␮LofPierceSuperblockbuffer(ThermoFischer Scientiﬁc,Melbourne,Australia).Nexteitherthestandards,seri- allydilutedfrom10to0.02ng/mL,orthetestairsamplesortest blankswereaddedtothewellsandincubatedfor3h.Afterwash- ing,the detection antibody dAb-␣TM diluted 1:500 in dilution buffer(PBS-Tcontaining1mg/mL BSA)wasaddedtothewells.

Subsequently,thestreptavidin-horseradishperoxidaseconjugate (Sigma–Aldrich,USA)diluted1:10,000indilutionbufferwasadded tothewellsandincubatedfor30min.Thewellswerethenwashed ﬁvetimesandpatteddry.Tovisualizeantibodybinding,100␮Lof 3,3,5,5-tetramethylbenzidinesubstrate(BectonDickinson,USA) wasaddedtothewellsuntilabluecolorationstartedformingin theblankwellsandthereactionstoppedusing1Nhydrochloric acid.Thecolordevelopmentinthewellswasmeasuredat450nm usingaVersamaxMicroplateReader(MolecularDevices,California, USA).

ValidationofsandwichELISA

Linearityofthestandardcurve,calculationoflimitofdetection (LOD)andlimitofquantitation(LOQ)andnon-speciﬁcity

The goodness of linearity of the rTm standard curve was assessedusingtheR²valuebasedonafour-parameterlogisticcurve calculatedusing SoftMax Prosoftware v5.2(Molecular Devices,

California,USA).Non-speciﬁcbindingoftheassaywasanalyzedby omittingthecaptureantibody,cAb-␣TMorthedetectionantibody, dAb-␣TM.TMlevelsintheaircollectionsampleswerederivedby interpolationoftheabsorbancereadingsoftherTmstandardcurve usingfourparametriclogisticalgorithm.Theaircollectionsamples weredilutedintherangeof1:2–1:80toobtainanabsorbancevalue withinthelinearrangeofthestandardcurve.Theallergenstandard curvewasincludedinevery96wellplateusedtoanalyzethetest samples.

Totestthesensitivityofthisassay,blanksampleswererunin 12separateexperimentsintriplicates.Thelimitofdetection(LOD) wascalculatedasthemeanoftheblanksamplesplusthreetimes thestandarddeviation.Thelimitofquantitation(LOQ)wascalcu- latedasthemeanoftheblanksamplesplustentimesthestandard deviation(Armbrusteretal.,1994).

Spikerecoveryassays

Spikingtestswereperformedtotestthematrixinterference effects of the extractionbuffer and other extraneousair-borne entitiesintheactualairsample.Seventeenrandomair-collection sampleswereselectedwitheitherlowlevelsoftropomyosinor withlevelsbelowtheLOQ,andspikedwithrTm.Anequalvolume oftestsamplesandrTm(spike)weremixedtoaﬁnalconcentra- tionof1ng/mL.Thesespikedsampleswerethenfrozenat−80^◦C overnighttosimulatetheaircollectionsamplepreparationprocess.

Thenextday,thespikedsampleswerethawedtoroomtemper- atureandtestedusingtheimmunoassayasdescribedinSection

“Assayprocedure”.Percentrecovery(%)wasderivedbydividing themeasuredTMconcentrationofthespikedsamplebythesum ofTMconcentrationofun-spikedsampleandthespikeconcentra- tion;[spikedsample(ng/mL)/(un-spikedsample(ng/mL)+Spike, 1ng/mL)]. The recoveryrates of the sample had tofall within 70–110%fortheassaytopass(Lexmondetal.,2011).

PrecisionoftheELISA

TheprecisionofthissandwichELISAwastestedonthebasis ofintra-assayandinter-assayvariability.Air-collectiontestsam- ples with tropomyosin levels below the LOQ were pooled and diluted1:2inextractionbuffertobespiked. Threespikedsam- pleswerepreparedatlow (0.2ng/mL),medium(0.5ng/mL)and high(1.0ng/mL)concentrationsofrTm.For intra-assayvariabilitytests,9 replicatesweretestedfor each spikeconcentrations (low, medium and high) in one single 96 well plate which includedastandardcurve.Totesttheinter-assayvariability,the 3spikedsamplesweretested insix differentexperimentsover threedaysbytwoindependentoperators.Eachtestsamplewas tested in triplicates and each experiment included a standard curve.For the assaytopass, theco-efﬁcient ofvariation (%CV) ofthereplicateshadtofallwithin20%,forbothintra-assayand inter-assay tests. In addition, the percent recovery had to fall within20%ofthetheoreticalconcentration(Kelleyand DeSilva, 2007).

Statisticalanalysis

TheMann–WhitneyUTestwasusedtocomparetheallergen exposurelevelsineachcategoryofwork-tasks.Apvalueofless than0.05wasconsideredsigniﬁcant.Allstatisticalanalysiswas performedusingGraphPadPrismversion6.02(GraphPad,USA).

Results

Characterizationoftheallergenstandard(rTm)

Theallergenstandard,rTmwassuccessfullyexpressedinaBL21 E.coli bacterialexpressionsystemasrepresentedin Fig.1.rTm

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Fig.1.Schematicrepresentationofthemethodologyandsetupusedforthedetectionandquantiﬁcationoftheair-borneshellﬁshallergentropomyosin.

Fig.2.Characterizationoftheimmunoassayallergenstandard,recombinanttropomyosin,rTm.(A)ProteinpuriﬁcationproﬁleofrTmusingsizeexclusionchromatography.

(B)SDS-PAGEanalysisofthevariouspuriﬁcationstagesoftheallergenstandardusingnickelafﬁnity(IMAC),sizeexclusionchromatography(SEC)andIgEimmunoblotting analysisofrTmusingpooledpatientsera.(C)Threedimensionalhomologymodelofcrustaceantropomyosinindimericform.(D)Comparisonofalpha-helicalcontentof allergenstandard,rTm(green)andnaturaltropomyosin,nTM(orange)usingCDspectroscopy.(Forinterpretationofthereferencestocolorintext,thereaderisreferredto thewebversionofthisarticle.)

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wasexpressedasafusionproteinwitha6XHistidine-tagatthe N-terminal end of the proteinto facilitate purification.Affinity chromatographywasperformedtopurifyrTmfromthecrudebac- teriallysate.However,severaladditionalbandscouldbeobserved in theaffinity purified fraction(Fig.2A).Therefore, size exclu- sion chromatography (SEC) was subsequently performed as an additionalpurificationstep.Thefinalproductwasvisibleasasin- glebandofapproximately40kDa.Todemonstrateimmunological reactivity,immunoblottingexperimentswereperformed(Fig.2B).

ImmunoblottingwithpatientseraconﬁrmedIgEantibodyreactiv- ityandthustheallergenicityofrTm.Proteinhomologymodeling of theallergen tropomyosin representsits highlystable alpha- helicalstructureandthefavorableformationofahomo-dimeric state(Fig.2C).

CDspectroscopicanalysisofrTmandnaturaltropomyosinwas performed,toconﬁrmappropriateproteinfoldingand structure oftherecombinantproteinanditssubsequentuseasanallergen standard(Fig.2D).Adistinctnegativesignalat208and222nmis typicalforanalphahelicalprotein.Thiswasalsoobservedforthe rTmascomparedtothepuriﬁednaturaltropomyosin.TheCDspec- trumforrTmwasalmostidenticaltothatofnaturaltropomyosin.

Bindingpropertiesofthecaptureantibody

Thepolyclonalanti-tropomyosinantibody,cAb-␣TMwassuc- cessfullyisolatedfromtheIgGenrichedfractionoftheimmunized rabbitserumasshowninFig.1.Immunoblottingdemonstratedspe- cificbindingofcAb-␣TMtoa37kDabandfromprawncrudeextract (Fig.3A).Specificantibodyreactivitytotropomyosinwasconfirmed withstrongbindingtorTm(Fig.3B).Moreover,antibodyreactiv- itywasobservedtothedimericformoftropomyosinformedat 75kDa.TheantibodybindingcharacteristicsofcAb-␣TMwasana- lyzedagainstvariousantigenicsources.cAb-␣TMshowedstrong bindingtotropomyosinfromthecrustaceansanalyzed;crab,prawn andlobster.Ontheotherhand,nobindingwasobservedtoextracts fromfish,chickenandE.coli(Fig.3C).Interestingly,antibodybind- ingwasobserved totropomyosin fromhousedustmite extract at37kDaregion.Thus,antibodyspecificitywasdemonstratedto invertebratetropomyosin.

Selective antibody binding to crustacean tropomyosin may beattributedto moleculardifferences inthe primary structure of tropomyosin among vertebrates and invertebrates (Fig. 4A).

Thecompared crustaceantropomyosinwasat least94%identi- calamong each other.However, when compared tovertebrate tropomyosin,themaximumpercentidentitywasonly58%(Fig.4B).

Cockroachandhousedustmitetropomyosin,bothofwhichhave beencharacterizedasallergens,werecloselyrelatedtocrustacean tropomyosinwith79–82%sequenceidentity(Fig.4BandC).

Linearityofallergenstandardcurve

AtenpointserialdilutioncurveofrTmwasusedintherange of0.02–10ng/mLconcentration,dilutedusingairsampleextrac- tionbuffer. Astandard curvewasgenerated usingtheabsolute allergenconcentrationsinng/mLanditscorrespondingabsorbance valuesmeasuredat450nmusingafour-parameterlogisticregres- sionalgorithm. Errorbarsrepresentthestandarddeviation.The goodnessofﬁt(R²)was0.998averagedfromsixindividualexper- iments(n=6)(Fig.5A).Thelinearregionofthestandardcurve, 0.02–1.25ng/mLwasusedforthequantiﬁcationoftropomyosin intheaircollectionsamples.

Toquantifytropomyosinintheaircollectionsamplestheyhad tobedilutedfromatleast1/2to1/80tobringtheabsorbanceval- ueswithinthelinearrangeofthestandardcurve.Thisallowedfor accuratemeasurementofsampleswithveryhighorlowallergen content.UseofhigherconcentrationsofrTm,beyond2.5ng/mL,

Table1

MatrixinterferenceandspikerecoveryanalysisoftheTmsandwichELISA.Spike recoveryassaywasperformedon17randomaircollectionsamplesbyspikingwith 0.5ng/mLofrTmstandard.%recoverywascalculatedbycomparingthetheoreti- calconcentrationanddetectedconcentrationofrTminng/mL.Meanrecoverywas calculatedas78.8±6.5%.

Sampleno. Unspiked air-collection samples

Spikedsamples withrTm (0.5ng/mL)

Recoveryof spikeTm

Tm(ng/mL) Tm(ng/mL) Percent(%)

1 <LOQ 0.36 71.0

2 <LOQ 0.39 78.0

3 <LOQ 0.40 80.0

4 <LOQ 0.34 67.6

5 <LOQ 0.43 86.0

6 <LOQ 0.41 81.2

7 <LOQ 0.36 71.6

8 <LOQ 0.40 79.0

9 <LOQ 0.34 68.4

10 <LOQ 0.46 91.8

11 <LOQ 0.40 79.4

12 0.69 0.94 78.9

13 0.31 0.64 79.6

14 0.36 0.71 83.2

15 0.89 1.24 89.2

16 0.29 0.60 76.2

17 0.49 0.78 79.0

did not result in a proportional increase in the absorbance values(Fig.5B).Thelimitofdetection(LOD)wascalculatedtobe 60pg/mLandthelimitofquantitation(LOQ)wasdeterminedtobe 100pg/mL.

Assayspeciﬁcity

Theaccuracyofanimmunoassaydependsontheabsenceof non-specificantibodybinding.Thiscanbeattributedtospecific non-specificbindingandnon-specific,non-specificbinding(Kelley andDeSilva,2007).WetestedcAb-␣TMforspecific,non-specific bindingandasshowninFig.3nobindingwasobservedtoanyother proteinbuttropomyosin.Subsequently,weperformedtheassay withtheentirestandardcurvebyomittingthecaptureantibody, cAb-␣TMorthedetectionantibody.Thisconfirmedthespecificity ofthisassaytorTmwithabsenceofnon-specificbindingtoother reagents,sinceacompletelossofsignalwasobserved(Fig.5A).

Finally,weperformedthespikerecoverytesttoanalyzetheinter- ferencefromnon-relatedmatrixagentspresentintheair-borne particulatemattercollectedintheairsamples(Table1).Resultsare shownaspercentrecoveryoftherTmspike(0.5ng/mL)inblank samplesand test sampleswithtropomyosin contentbelowthe assayLOQ.Themeanrecoveryofthespikewascalculatedtobe 78.8±6.5%whichpassedourpre-setcriteria(KelleyandDeSilva, 2007;Leeetal.,2006;LeeandHall,2009).Moreover,therewere nocasesofpositiveinterferenceamongcontrolsamples.Twoof17 sampleshadarecoverybelow70%suggestingpossibleinterference withthesamplematrix.

Intra-assayandinter-assayvariability

Intra-andinter-assayvariabilitytestswereconductedtotestthe precisionandaccuracyoftheassay(Table2).Variabilitywastested forlow,mediumandhighconcentrationoftropomyosin(Fig.5C).

Themeanrecoveriesofthesampleswereover70%andtheco- efﬁcientofvariationwas<20%forallthreeconcentrationsofrTm tested.Thisdataareinconcordancewiththeacceptancecriteria forassayvalidation(KelleyandDeSilva,2007;Leeetal.,2006;Lee andHall,2009).

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Fig.3.Bindingcharacteristicsofpolyclonalrabbitanti-tropomyosinIgGantibody.(A)Antibodybindingpatternsofvariousstagesofrabbitantibodypuriﬁcationagainst prawnproteinextract.(B)SDS-PAGEandimmunoblottingoftheallergenstandardrTmusingcAb-␣TMantibody.(C)SpeciﬁcantibodybindingofcAb-␣TMagainstdifferent proteinextractsofinvertebrateandvertebratespecies.

Analysisofairsamplesfromcrabprocessingfactory

Air-samplesweretested fromthePBZsofworkersfromtwo differentprocessingactivities;kingcrabandediblecrab(Table3).

Theaverageairvolumesampledwas1095±118Land830±371L forediblecrabandkingcrabprocessing,respectively.Theamount ofair-bornetropomyosinandexposurepatternsdifferedamong thetwoprocessingplants(Fig.6).Tropomyosininthekingcrab plantmeasuredintherangeof0.15–75.89ng/m³ whereasinthe ediblecrab,itwas0.42–138ng/m³.

Intheedible crabprocessing,highest tropomyosin exposure wasdemonstrated for workers handlingboiledmeat and spin- celler(separatorofmeatfromboiledcrab).Thelowestexposure

was in the scanning process, freezer and raw crab handling area.Thetropomyosinexposureconcentrationsvariedsigniﬁcantly among workersinthehighexposureactivities,handlingboiled crab.

Kingcrabprocessing wasperformedin batchesinterspersed withﬁshprocessing,duetotheseasonalavailabilityofthecrab.

Thehighesttropomyosinconcentrationswereidentifiedforpro- cessessuchascleaning,crackingandcrabdegilling.Amongworkers sharingworktasks betweencrabandfishprocessing,moderate concentrationsoftropomyosinexposurewasobserved.Locations and taskssuchastruck driving,fishpacking,receivingstations and fishgutting didnotshow anysignificantconcentrationsof air-bornetropomyosin.

Table2

Assayprecisionoftheimmunoassay.Intra-andInter-assayvariabilityoftheassaywastestedusing0.2ng/mL,0.5ng/mLand1ng/mLofrTmaslow,mediumandhighantigen concentrations,respectively.Theassayvariabilityisshownintermsofthecalculatedmeanconcentration,standarddeviationandpercentageofthecoefﬁcientofvariation.

RecoveryoftherTmconcentrationswerecalculatedasapercentageoftheratioofcalculatedversustheoreticalconcentrations.Numberofreplicates;n=9(intra-assay)and n=6(inter-assay).

rTm(ng/mL) Mean(ng/mL) Standarddeviation,SD Coefﬁcientofvariation,CV(%) Recovery(%)

Intra-assayvariability

0.2 0.14 0.013 9 70.5

0.5 0.40 0.025 6.3 79.8

1.0 0.76 0.074 9.8 76.4

Inter-assayvariability

0.2 0.16 0.018 11.0 80.0

0.5 0.41 0.043 10.4 82.0

1.0 0.81 0.074 9.1 81.0

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Fig.4.Comparisonofinvertebratetropomyosinprimarystructure.(A)Aminoacidsequencealignment,forvisualcomparisonofprimarystructuresimilarityofinvertebrate andvertebratetropomyosins.Accessionnumber(Genbanknucleotidesequencedatabase)Prawn,Penaeusmonodon,accessionnumber(AAX37288.1);Crab,Portunuspelagicus (BAF47266.1);Lobster,Jasuslalandii(AFY98827.1);Housedustmite,Dermatophagoidespteronyssinus(ACI32128.1);Cockroach,Blatellagermanica,(AAF72534.1);Fish,Gadus chalcogrammus(BAC44994.1);andPig,Susscrofatropomyosin(NP001090952.1).Thecoloredshadingintensityindicatespercentidentity.(B)Tropomyosinaminoacid sequenceidentitytablecalculatedusingClustalOmega.(C)Phylogenetictreeofinvertebratetropomyosininferredusingtheminimumevolutionmethod.

Discussion

Frequentoccurrencesofallergicreactionsamongseafoodwork- ersduetoair-borneallergenexposurehavebeenreported(Lopata andJeebhay,2013).Occupationalasthmahasbeencommonlyasso- ciatedwithshellfishprocessingandpreviousstudieshaveshowna prevalenceofupto36%(JeebhayandCartier,2010).Astrongcor- relationbetweenthehighair-borneallergenconcentrationsand developmentofallergenicsensitizationandasthmahasbeensug- gested(Brisman,2002;JeebhayandCartier,2010).Anumberof tasksintheshellfishprocessingworkplaceputtheworkersata greaterriskofexposureandconsequentsensitizationtoshellfish

allergens(Cartier,2010;LopataandJeebhay,2013).Currently,there isa lackof standardized,validated methodsfor monitoringthe allergenconcentrationsinbioaerosolsproducedduringshellﬁsh processing.

Theaimofthisstudywastodevelopandvalidateasensitive antibody-basedimmunoassayforthedetectionandquantiﬁcation oftheshellﬁshallergentropomyosininbioaerosolsproduceddur- ingcrabprocessing.

Traditionally, serum IgE antibodies from shellﬁsh-sensitized individuals have been used to detect air-borne allergens, using an inhibition ELISA setup (Malo et al., 1997; Weytjens et al., 1999). This approach is useful for quantifying air-borne

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Fig.5.Standardcurvefortheallergenstandard,recombinanttropomyosin(rTm).(A)A10pointserialdilutioncurvefrom10to0.02ng/mL,errorbarsindicatethestandard deviationofeachdilutionoversixindividualexperiments.(B)Assayreproducibilityandspeciﬁcity:TestAandBindicatethestandardcurvesfromtwoseparateexperiments.

“䊉”and“X”indicateomissionofthecaptureordetectionantibodiesrespectivelytoanalyzenon-speciﬁcbindingpropertiesoftheimmunoassay.(C)Inter-assayvariability testusingrTmspikedsamplesatthreedifferentconcentrations;0.2,0.5and1.0ng/mL(n=6).

IgE-reactiveallergens,withtheassayhavingasensitivedetection limit of 1ng/mL. However,the majordisadvantage ofIgE antibodies istheirlow titerand difﬁcultyindeveloping a standard assaymodelduetovariedantibodyreactivityindifferentpatient sera.

ELISAbasedimmunoassayshavebeenpreviouslyappliedfor thedetectionoftropomyosininfoodmatrices,usingmonoclonal orpolyclonalantibodybasedplatforms(Jeoungetal.,1997;Seiki etal.,2007;Werneretal.,2007).However,theassaysensitivity, precisionandmatrixinterferencelevelsvariedlargelyamongthe differentassays.Monoclonalantibodybasedassayshavetheadvan- tageofhighspeciﬁcityandlackoffalse-positiveresultsinallergen detection(Lopataetal.,2002,2005).Nevertheless,anymodiﬁca- tionsorchangesintheconformationofthemAb-epitopeonthe allergenic protein maylead to lossof binding. Polyclonal anti- bodybasedassaysontheotherhand,canbindtotheallergensat

multipleepitopes,thusminimizingtheriskofproteinconforma- tionalchangesaffectingthebindingtotheallergen.

Inthepresentstudy,wehaveemployedamodifiedapproachfor developingahighlysensitiveimmunoassayforthequantification ofaerosolizedcrustaceanallergens.Oneoftheinitialhurdleswasto developapolyclonalantibodybasedassaywithhighspecificityand minimizedunspecificbinding.Naturaltropomyosinspurifiedfrom fourdifferentcrustaceanspecieswereusedforthegenerationof polyclonalrabbitantibodies.Thisincreasedthebindingcapacityof theantibodytotropomyosinswithanaminoacidsequencevaria- tionofover95%.Moreover,arecombinantcrustaceantropomyosin ofhighpuritywasusedasthestationaryphaseforaffinitypurifica- tionoftropomyosin-specificIgGantibodiesfromtherabbitserum.

Theresultantfinalantibodyfractiondemonstratedhighspecificity tocrustaceantropomyosinwithnonon-specificbindingtoother homologousproteinsasshowninourvalidationtests.

Table3

Exposuretoair-borneallergentropomyosinamongworkersincrab-processingworkplaceaccordingtospeciﬁctasksperformed.

Designation Taskfunction Numberofsamples Allergenexposure pValue^*

Median(ng/m³) Range(ng/m³)

A Handlingboiledmeat 9 61.4 21.95–138.8 <0.0001

B Crabcracking 22 23.48 <LOQ-75.89 <0.0001

Gutting

Rawmeathandling Cutting

Cleaning

C Receivingstation 17 1.11 <LOQ-71.92 <0.0016

Scanning Sorting Packing

D Transport/logistics 27 0.25 <LOQ-6.74 –

Fish-relatedtasks

*PvalueswerecalculatedusingtheMann–WhitneyUtestincomparisontocategoryD.

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Fig.6. Allergenexposuretocrabprocessingworkersinvolvedinvarioustasksand locations.Allergenexposuretoworkersprocessingediblecrabsisdisplayedasblue dotsandkingcrabsinreddots.Themeanexposureforeachtaskcategoryisshown asahorizontallineinblueandredforediblecrabandkingcrab,respectively.Tasks:

(A)Handlingboiledmeat,(B)crabcracking,gutting,rawmeat,cuttingandcleaning, (C)crabreceivingstation,handling,scanning,sortingandpacking,and(D)logistics andﬁsh-relatedtasks.(Forinterpretationofthereferencestocolorintext,thereader isreferredtothewebversionofthisarticle.)

Interestingly,thecaptureantibodywasabletorecognizehouse dustmitetropomyosin,duetothelatter’shighpercentidentityto crustaceantropomyosin.Thishowevermaynotsigniﬁcantlyaffect thequantiﬁcationofcrabtropomyosininawetprocessingenviron- ment.Previousstudieshaveshownthatdustmitescontainverylow amountsoftropomyosin(Arlianetal.,2009).Moreover,ithasbeen shownthattropomyosinmaynotbethemainallergeninvolvedin seafood-mitesensitization(Boqueteetal.,2011).

Toimprovetheassayspeciﬁcity,recombinanttropomyosinwas usedasanallergenstandardintheassaytoquantifytheamountof tropomyosininthesamples.Theadvantageofusingarecombinant protein as a standard is the unlimited availability and consis- tentperformanceasopposed tothenatural sourcewhichoften demonstratesbatchtobatchvariations.Previousstudiesonaller- gendetection have developed assays withsensitivities ranging from1ng/m³to105ng/m³(Lopataetal.,2005;Maloetal.,1997).

Theimmunoassaydevelopedinthisstudywasabletoachievean allergendetectionlimitof60pg/m³.However,assayswithhigher sensitivity(10pg/m³)havebeendevelopedformouseorraturinary allergens(Renstrometal.,2002).

Themainparametersofperformanceoftheassaywereestab- lished by our in-house validation. Spike recovery tests were performedto test the matrix interferenceeffects and recorded as79%.Whiletheoutcomefellwithintheacceptancecriteria,it highlightedtheeffectsofmatrixcomponentsonallergen–antibody binding.Thisinterferenceseemstoresultmainlyfromtheextrac- tionbufferusedtoelutetheallergensfromairsamplingﬁlters.

Theperformanceoftheallergenstandardcurvewastestedinthe presenceandabsenceofthebufferandshowedamarkedeffecton theabsorbancevaluesoftheallergenstandard(datanotshown).

Therefore, tomaintainsimilarlevelsof matrix effects fromthe bufferonthetestsamplesaswellasthestandards,thelaterwas dilutedusingthesameextractionbufferasforthesampleanaly- sis.Theaccuracyandprecision(reproducibility)oftheassaywas testedusinginter-assayandintra-assayvariabilitytests.Boththe testsmettheacceptancecriteriaoffallingwithin20%co-efﬁcientof variation.

Thisdeveloped immunoassaywasutilizedtoassessthecon- centrationsofaerosolizedtropomyosinintwodifferentprocessing activities; edible crab and king crab. In general, the levels of bioaerosolsand subsequently the air-borneallergen concentra- tionsaredependentonvariablessuchasthekindofseafoodbeing processed,amount of seafood beingprocessed, size of thefac- tory,layoutofprocessingequipmentandlocationsofventilation

system.Thesevariablesvarywithdifferentlocationsanddifferent seasonsofseafoodprocessing.Forexample,inthisstudy,someking crabprocessingfactoriesweremainlyinvolvedinfishprocessing andonlytemporarilyconvertedtocrabprocessing,dependingon theavailability and fishing season.Due tothis, a small lineof crabprocessingassembly wasplacedinrelativelylargeproduc- tionroomswhereventilationmaynothavebeenoptimalnearthe processingactivity.Someoftheediblecrabprocessingactivities involvedcookingthecrabs.Thiswasperformedindefinedloca- tionsorseparateroomswithpointventilations.Althoughmeasures weretakentolimittheexposuretothefumesfromthecooking vats,manyworkersnotinvolvedinthecookingactivitiesweresta- tionedclosebyandwereexposedtothefumes.Theediblecrab processingfacilitywasdesignedspecificallyforcrabproduction anditinvolvedworkersstandingclosetogetheronaprocessing line.Inthekingcrabfactories,thelargerroomsandfewerworkers involvedinprocessingandmoremanuallabormightaccountfor someofthedifferencesinallergenconcentrationsintheairsamples ascomparedtotheediblecrabfactory.

Several shellfish allergens have been identified and charac- terizedincommonlyconsumed shellfishspecies (Kamathetal., 2013,2014;Lopataetal.,2010).Tropomyosin,howeveristhemost abundantandheat-stableinvertebrateallergencapableofinduc- ingallergic sensitization.In this study,thehighestexposureto tropomyosinwasdemonstratedduringheatingandboilingpro- cesses as shown previously (Malo et al., 1997).Recent studies haveshowntheabilityofnaturalorheatgeneratedtropomyosin fragmentstoelicitIgEreactivity.Moreover,enhancedIgEreactiv- ityoftropomyosinafterheatingwasdemonstrated(Abramovitch etal., 2013;Kamathetal., 2013).Thisis ofclinicalimportance sincetheheatingprocessesdonotonlyincreasetheaerosolization ofallergensbutcanalsoexacerbatetheIgEsensitizationamong affectedworkers.Fromthedetectionpoint-of-view,itisanadvan- tagetoemployapolyclonalantibodybasedassay,sincemonoclonal basedassaysmaynotbeabletodetecttheseallergenfragments aerosolizedduringtheheatingorboilingprocesses(Kamathetal., 2013).Theimportanceofallergenfragmentsisparticularlyimpor- tantinthecaseoftropomyosin,aseightIgEbindingepitopeshave beendiscoveredspanningitsentirelengthofthealpha-helicalsec- ondarystructure(Kamathetal.,2013;Lopataetal.,2010;Reese etal., 2005).Thismaydramaticallyincrease possibleIgEcross- linkinginsensitizedindividualsduetoexposuretotropomyosin fragments.

Insummary,wehavedevelopedandvalidatedahighlysensi- tiveassayforair-bornetropomyosindetection.Usingthisassay, wewereabletoquantifytropomyosininthePBZofworkersper- formingdifferentworkactivitiesincrabprocessingfactories.High concentrationsoftropomyosin weredetectedmainlyin boiling, heating and de-gilling stations. Thedeveloped immunoassay is currentlyemployedformonitoringallergenexposureindifferent crabprocessingfactoriesaspartofabiggerwork-safetystudyin Norway.The methodologicalapproach usedfor developing this assayoffersopportunitiestootherfoodindustriestomonitorair- borneallergens,soastoimprovework-safetyand occupational health.

Acknowledgements

This study received ﬁnancial support from the Norwegian Asthma and AllergyAssociation witha grant from the Norwe- gianExtraFoundationforHealthandRehabilitationthroughEXTRA funds.ALisholderofAustralianResearchCouncil–FutureFellow- ship.PatientserawerekindlyprovidedbyRobynE.O’Hehirand Jennifer Rolland, The Alfred Hospital, Prahran, Melbourne, VIC, Australia.