immunological reactivity using monoclonal and polyclonal...

Research ArticleImmunological Reactivity Using Monoclonal andPolyclonal Antibodies of Autoimmune Thyroid Target Sites withDietary Proteins

Datis Kharrazian123 Martha Herbert12 and Aristo Vojdani34

1Harvard Medical School Boston MA USA2TRANSCEND Research Department of Neurology Massachusetts General Hospital Charlestown MA 02129 USA3Department of Preventive Medicine Loma Linda University School of Medicine Loma Linda CA USA4Immunosciences Laboratory Inc Los Angeles CA USA

Correspondence should be addressed to Datis Kharrazian datis_kharrazianhmsharvardedu

Received 10 April 2017 Revised 14 June 2017 Accepted 15 June 2017 Published 15 August 2017

Academic Editor Marian Ludgate

Copyright copy 2017 Datis Kharrazian et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Many hypothyroid and autoimmune thyroid patients experience reactions with specific foods Additionally food interactions mayplay a role in a subset of individuals who have difficulty finding a suitable thyroid hormone dosage Our study was designed toinvestigate the potential role of dietary protein immune reactivity with thyroid hormones and thyroid axis target sitesWe identifiedimmune reactivity between dietary proteins and target sites on the thyroid axis that includes thyroid hormones thyroid receptorsenzymes and transport proteinsWe alsomeasured immune reactivity of either target specificmonoclonal or polyclonal antibodiesfor thyroid-stimulating hormone (TSH) receptor 51015840deiodinase thyroid peroxidase thyroglobulin thyroxine-binding globulinthyroxine and triiodothyronine against 204 purified dietary proteins commonly consumed in cooked and raw forms Dietaryprotein determinants included unmodified (raw) and modified (cooked and roasted) foods herbs spices food gums brewedbeverages and additives There were no dietary protein immune reactions with TSH receptor thyroid peroxidase and thyroxine-binding globulin However specific antigen-antibody immune reactivity was identified with several purified food proteins withtriiodothyronine thyroxine thyroglobulin and 51015840deiodinase Laboratory analysis of immunological cross-reactivity betweenthyroid target sites and dietary proteins is the initial step necessary in determining whether dietary proteins may play a potentialimmunoreactive role in autoimmune thyroid disease

1 Introduction

Immunologic cross-reactivity occurs when adaptive immuneresponse against one antigen also occurs to another antigenwith amino acid structural similarity Immunological cross-reactivity was first identified in 1942 when it was found thatindividuals sensitized to pollen allergens developed immunereactivity to specific fruits [1] Further study found that cross-reactivity with pollen could also occur to human tissue targetproteins [2]

Exposure to antigens that share amino acid sequencehomology with self-tissue proteins in susceptible hosts has

been theorized as a trigger for tissue-specific autoimmunedisease [3 4]

Various antigens have been shown to specifically cross-react with thyroid tissue and trigger thyroid autoimmunityAdditionally heat shock protein 60 (Hsp60) a mitochon-drial chaperonin involved in stress responses diabetes andimmunological disorder has structural similarity to thy-roglobulin and thyroid peroxidasemolecules Enzyme-linkedimmunosorbent assay (ELISA) evaluations have also shownimmunological cross-reactivity to play a role in Hashimotorsquosthyroiditis [5]

HindawiJournal of yroid ResearchVolume 2017 Article ID 4354723 13 pageshttpsdoiorg10115520174354723

2 Journal of Thyroid Research

Numerous gastrointestinal pathogens have demonstratedmolecular mimicry with thyroid tissue For example humanmonoclonal thyroid-stimulating hormone receptor (TSH-r)has been shown to cross-react with Yersinia enterocolitica(Y enterocolitica) thereby providing a mechanistic frame-work for molecular mimicry in Gravesrsquo disease where Yenterocolitica antibody production promotes cross-reactivepathogenic response to TSH receptor [6] Researchers havediscovered that the outer membrane of the porin F protein ofY enterocolitica shares cross-immunogenicity with a leucine-rich domain of TSH receptor and plays a role in inducingautoimmunity to TSH receptor through molecular mimicry[7]

Immune cross-reactivity between Helicobacter pylori (Hpylori) and autoimmune thyroid disease has been suspecteddue to correlations between early onset H pylori infectionsand Hashimotorsquos hypothyroidism and Gravesrsquo disease [8 9]Cross-reactivity with Candida albicans and thyroid antigenshas been identified and associated with the development ofautoimmune thyroid disease [10] The protozoa Toxoplasmagondii has also been reported to potentially induce autoim-mune thyroid cross-reactivity through molecular mimicrymechanisms [11]

In addition to gastrointestinal pathogens Clostridiumbotulinum neurotoxin A (Btx) has been reported to induceserum elevations of TSH Researchers found that Btx andthyroid autoantigens share amino acid sequence homologyandmay play a role in cross-reactive complication of autoim-mune thyroid disease [12] Borrelia burgdorferi has beenshown to have protein homology with TSH receptor andtherefore plays a role as an antigenic trigger for autoimmunethyroid disease [13] Coxsackie virus antibodies have alsodemonstrated cross-reactivity with the thyroid and have beenreported to be a contributing factor to the pathogenesis ofautoimmune thyroid disorder [14]

The list of pathogenic organisms such as viral bacterialfungal spirochete and protozoa antibodies that may con-tribute to tissue-specific thyroid autoimmunity via antibodyamino acid sequence homology is a growing field of studyHowever little research has been done with food proteinantibodies and their potential role in thyroid specific cross-reactivity Published research has looked at gluten and thyroidautoimmunity but the immunological reactive role that otherdietary proteins may play with thyroid function has been alimited area of research [15 16]

In this study we evaluated the potential for food cross-reactivity with the thyroid axis by evaluating immune reac-tivity between purified dietary proteins and thyroid targetspecific antibodiesWemeasured immune reactivity of eithertarget specific monoclonal or polyclonal antibodies for TSHreceptor 51015840deiodinase thyroid peroxidase thyroglobulinthyroxine-binding globulin thyroxine and triiodothyronineagainst 204 purified dietary proteins that are commonly con-sumed in raw or cooked forms Food determinants includedunmodified (raw) and modified (cooked and roasted) foodproteins herbs spices food gums brewed beverages andadditives We included raw versus modified food antigenssince in our earlier study we showed that some individualsmay react to raw food antigens but not heat-modified ones

while conversely others may react to heat-modified but notraw food antigens [17]

Specific target proteins along the thyroid axis associatedwith autoimmune thyroid disease were evaluated in our studyincluding thyroid-stimulating hormone receptor (TSH-R)51015840deiodinase thyroid peroxidase thyroglobulin thyroxine-binding globulin thyroxine and triiodothyronine TSH-Rantibody reactions are the hallmark of Gravesrsquo disease Theseantibody reactions promote autoimmune reactivity againstthe target protein but homosteric binding to these receptorscan promote increased production of thyroid hormonesleading to hyperthyroidism [18]Thyroid peroxidase (TPO) isthe key thyroid enzyme necessary for the synthesis of thyroidhormones and is the target for Hashimotorsquos autoimmunityTPO antibodies are associatedwith destruction of the thyroidand thyroiditis [19] Thyroglobulin (Tg) is a dimeric proteinsynthesized in the thyroid and used for production of thyroidhormones Its level is elevated with thyroid tissue breakdownsuch as with thyroiditis and differentiated thyroid cancer[20] Tg is a common target for thyroid autoimmunity [21]Thyroxine-binding globulin (TBG) is the transport proteinfor thyroid hormones in circulation and has high affinityfor thyroxine and triiodothyronine [22] Levels in the bodycan change due to metabolic disease pregnancy oral contra-ceptive use and hormone replacement therapy [23] Type IIiodothyronine deiodinase (DIO

2) is the enzyme that converts

prohormone thyroxine by outer ring deiodination to bioac-tive triiodothyronine [24] Thyroxine (T

4) is a prohormone

synthesized by the thyroid gland and composed of four iodinemolecules attached to thyroglobulin Triiodothyronine (T

3)

is bioactive thyroid hormone that is responsible for the keyphysiological mechanisms of thyroid target tissue function[25]

The identification of immunological food protein cross-reactivity in the laboratory with thyroid axis target sitesis the first step in understanding whether dietary proteinsmay potentially play an immunological reactive role inautoimmune thyroid disease In this study we attempt to takethe initial first step by determining any potential patternsof immunological cross-reactivity with a diverse list of foodproteins an specific thyroid axis autoimmune target sites

2 Materials and Methods

21 Polyclonal and Monoclonal Antibodies Affinity-purifiedrabbit polyclonal thyroid-stimulating hormone receptor anti-body affinity-purified goat polyclonal thyroxine 5-deiodinase(DIO2) antibody monoclonal antibody to thyroid perox-

idase monoclonal antibody against thyroglobulin mousemonoclonal thyroxine antibodymousemonoclonal antibodyto triiodothyronine and monoclonal antibody to thyroxine-binding globulin were purchased from MyBioSource Inc(San Diego CA USA)

22 Preparation of Dietary Antigens Food antigens wereprepared from products purchased from the supermarket inboth raw roasted or cooked form For that preparation 10 gof food product was put in a food processor using 01M ofphosphate buffer saline (PBS) at pH 74Themixer was turned

Journal of Thyroid Research 3

on and off for 1 hour and then kept on the stirrer overnightat 4∘C After centrifugation at 20000119892 for 15 minutes thetop layer which contained oil bodies was discarded Theliquid phase was removed and dialyzed against 001M ofPBS using dialysis bags with a cutoff of 6000 kDa Dialysiswas repeated three times to ensure all small molecules wereremoved After dialysis all samples were filtered through a02micron filter to remove any debris Protein concentrationswere measured using a kit provided by Bio-Rad (HerculesCA USA) Different peptides were purchased from Bio-Synthesis (Lewisville TX USA) Lectin and agglutinins werepurchased from Sigma Aldrich (St Louis MO USA)

23 Preparation of Dietary Oleosin Antigens To purify theoleosin from peanuts corn safflower sunflower and soy-bean the foods were prepared according to the methoddescribed by Vojdani [26] A total of 100mL of chloro-formmethanol (21 vv) was then added and blended for 2minutes using a food processor The mixture was put in a50mL tube and centrifuged at 14000 RPM for 5minutesTheliquid in the upper phase was filtered through two layers offilter paper The resultant filtrate was collected in multipleglass bottles and dried under a stream of air with strongcontinuous agitation The chloroformmethanol extractionstep was repeated twice A total of 20mL of diethyl ether wasthen added and the white solid material stuck on the surfaceof the glass bottles was detached and resuspended in diethylether At this point 10mL of water was added to each bottlewhich was centrifuged at 20000119892 for 5 minutes The upperdiethyl ether layer that contained lipids was removed andthe white solid interface material containing the oleosinswas collected and transferred to microtubes with a minimumvolume of water and diethyl ether The microtubes were cen-trifuged at 20000119892 for 5minutesThe interfacialmaterial wasexposed to a stream of nitrogen to evaporate the remainingdiethyl ether One mL of chloroformethanol (955 vv) wasadded to the interfacial material in each tube The contentsof each tube were quickly vortexed and transferred to a glassflask To separate any protein contaminants from the oleosins10mL of chloroformmethanol (955 vv) was added and themixture was filtered through filter paper that was previouslyrinsed with chloroformmethanol The filtrate was collectedin a flask and dried under a stream of nitrogen The driedoleosins were dissolved in chloroformmethanol and appliedto a Sephadex LH-60 column (Bio-Rad Hercules CA USA)using chloroformmethanol as the solvent The collectedfractions of oleosins were checked by sodium dodecyl sulfate(SDS)-gel electrophoresis

24 Preparation of Dietary Gum Antigens Mastic gum car-rageenan xanthan gum guar gum gum tragacanth locustbean gum and 120573-glucan were purchased from Sigma Aldrich(Saint Louis MO USA) Extracts from these items wereprepared according to the procedures described by Vojdani[27] Ten grams of each gum was extracted in 500mL ofbuffer pH 46 by mixing them for 8 hours at 25∘C on amagnetic stirrer The solution was centrifuged at 20000119892and supernatant was removed and concentrated by a factorof 10 using an Amicon filter The protein concentration was

measured using a kit provided by Bio-Rad (Hercules CAUSA) All extracts were aliquoted and stored frozen at minus20∘Cuntil used Different gum extracts were dissolved in 01MPBS These antigens were diluted 1 50 in 01M carbonatebuffer pH 92 and 100 120583L of each gum antigen

25 Enzyme-Linked Immunosorbent Assay (ELISA) forDemonstration of Immune Reactivity Food antigensand peptides were dissolved in PBS or methanol at aconcentration of 10mgmL and then diluted 1 100 in 01Mcarbonate-bicarbonate buffer at a pH of 95 and 100 120583L wasadded to each well of the polystyrene flat-bottom ELISAplate Plates were incubated overnight at 4∘C and thenwashed three times with 200 120583L Tris-buffered Saline (TBS)containing 005 Tween 20 at a pH of 74 The nonspecificbinding of immunoglobulins was prevented by adding amixture of 2 bovine serum albumin (BSA) into the TBSand then incubating overnight at 4∘C Plates were washed asdescribed above and then serum samples diluted 1 100 in01M PBS Tween containing 2 BSAwere added to duplicatewells and incubated for 1 hour at room temperature

Plates were washed again and then polyclonal and mon-oclonal antibodies diluted at an optimal dilution of 1 500were added to duplicate antigen-coated wells plates wereincubated for an additional 1 hour at room temperatureThe plates were then washed five times with TBS-Tweenbuffer The enzyme reaction was started by adding 100 120583L ofparanitrophenylphosphate in 01mL diethanolamine buffer1mgmL containing 1mMMgCl

2and sodium aside at a pH

of 98 The reaction was stopped 45 minutes later with 50120583Lof 1N NaOH and the samples were read by an ELISA readerand the optical densities (OD) were recorded

For the determination of specificity of monoclonal andaffinity-purified polyclonal antibodies in reaction with var-ious food antigens four wells of each 96-well plate werecoated with 2 BSA alone but not the food antigens Afterthe addition of all other antigens the mean OD of these wellswere subtracted from all other reactions

26 Determination of Immune Reactivity Scale Two hundredand four proteins (see Table 1) were tested for seven target tis-sue antibodies in duplicate leading to 2856 antigen-antibodyOD measurements The results of each duplicate OD wereaveraged together for one OD value and the CD of controlwells which was less than 015 was subtracted from all othermeasurements Of the 2856 OD measurements the meanOD was 033 with a standard deviation (SD) of 053 The ODof 087 represented two standard deviations from the meanthe OD of 141 represented three standard deviations from themean and anODof 195 represented four standard deviationsfrom the mean OD values below two standard deviations orless than 053 were labeled nonsignificant OD values abovetwo standard deviations but below three standard deviationsat OD values of 066ndash086 were categorized as 1+ reactionOD values above three standard deviations but less thanfour standard deviations at OD values of 087ndash107 werecategorized as 2+ reactions OD values above four standarddeviations with OD values greater than 108 were categorizedas 3+ reactions (see Table 2)


Table 1 Dietary proteins screened for immune reaction

DAIRY and eggs modifiedAlpha-casein and beta-caseinCowrsquos milkChocolate milkEgg White boiledEgg Yolk boiledGoatrsquos milkMilk butyrophilinSoft cheese + hard cheeseWhey proteinYogurtGRAINS raw and modifiedAmaranthBuckwheatCasomorphinOatsQuinoaRiceRice white + brown boiledRice cakeRice proteinRice endochitinaseRye Barley Spelt Polish WheatSesameSorghumTapiocaTeffWild rice boiledWheat + alpha-gliadinsYeast millerHempBEANS modifiedBlack bean boiledBean agglutininsDark chocolate + cocoaFava bean boiledGarbanzo bean boiledKidney bean boiledLentil boiledLentil lectinLima bean boiledPinto bean boiledSoybean agglutininSoybean oleosin + AquaporinSoy sauce gluten-freeTofuNuts and seeds raw and modifiedAlmondAlmond roastedBrazil nut raw + roastedCashewCashew roastedCashew vicilinChia seedFlax seedHazelnut raw + roastedMacadamia nut raw + roasted

Table 1 Continued

Mustard seedPecan raw + roastedPeanut roastedPeanut butterPeanut agglutininPeanut oleosinPistachio raw + roastedPumpkin seeds roastedSesame albuminSesame oleosinSunflower seeds roastedWalnutVegetables raw and modifiedArtichoke boiledAsparagusAsparagus boiledBeet boiledBell PepperBroccoliBroccoli boiledBrussels sprouts boiledCabbage red + greenCabbage boiledCanola oleosinCarrotCarrot boiledCauliflower boiledCeleryChili pepperCorn + aquaporin boiledPopped cornCorn oleosinCucumber pickledEggplant boiledGarlicGarlic boiledGreen bean boiledLettuceMushroom raw + boiledOkra boiledOlive green + black pickledOnion + scallionOnion + scallion boiledPea boiledPea ProteinPea LectinPotato white bakedPotato white friedPumpkin + squash boiledRadishSafflower + sunflower OleosinSeaweedSpinach + aquaporinTomato + aquaporinTomato pasteYam + sweet potato bakedZucchini boiled


Table 1 Continued

Fruit raw and modifiedAppleApple ciderApricotAvocadoBananaBanana boiledLatex heveinBlueberryCantaloupe + honeydew melonCherryCoconut meat + waterCranberryDateFigGrape red + greenRed wineWhite wineGrapefruitKiwiLemon + limeMangoOrangeOrange juicePapayaPeach + nectarinePearPineapplePineapple bromelainPlumPomegranateStrawberryWatermelonFish and seafood raw and modifiedCod bakedHalibut bakedMackerel bakedRed Snapper bakedSalmonSalmon bakedSardine + anchovy cookedSea Bass searedTilapia bakedTrout bakedTunaTuna searedWhitefish bakedCrab + lobster boiledImitation crabClam boiledOyster boiledScallops searedSquid (calamari) searedShrimp searedShrimp tropomyosinParvalbumin

Table 1 Continued

Meat modifiedBeef boiled mediumChicken boiledLamb bakedPork bakedTurkey bakedGelatinMeat glueHerbs rawBasilCilantroCuminDillGingerOreganoParsleyRosemaryThymeSpices rawCinnamonCloveMintNutmegPaprikaTurmeric (curcumin)VanillaGUMSCarrageenanGum guarGum tragacanthLocust bean gumMastic gum + gum ArabicXanthan gumBrewed beverages and additivesCoffee bean protein brewedInstant coffeeBlack tea brewedGreen tea brewedHoney raw + processedBeta-glucanFood coloring

27 Binding of Serially DilutedThyroid Antibody to Fixed Con-centrations of Various Food Antigens For the demonstrationof anti-thyroid antibodies binding to food antigens differentwells of microtiter plates were first coated with optimalconcentrations (10 120583gwell) of cashew roasted cashew latexhevein egg yolk cooked and seaweed After the completionof all steps necessary for coating the plates each monoclonalantibody in dilutions of 1 500ndash1 512000 was added to twodifferent rows of microtiter plates and after completion of allELISA steps the optical densities (ODs) were measured

28 Binding of Fixed Concentration of Antibody to SeriallyDiluted Food Antigens Different food antigens in a concen-tration of 400 120583gmL were serially diluted Microtiter plateswere coated with 003ndash40 120583g of each antigen in duplicaterows After the incubation and blocking steps the addition


Table 2 Immunoreactivity of monoclonal antibodies to T3 and T4with food proteins

Food protein T3

T4

Almond minus ++Almond roasted ++ +++Amaranth +++ +Avocado + +Barley ++ minus

Black bean cooked minus +Brazil nut raw amp roasted + +++Brussels sprouts cooked + ++Buckwheat +++ minus

Casein (120572 amp 120573) + minus

Cashew ++ +Cashew roasted ++ +++Cashew vicilin + +++Chocolate ++ minus

Clam cooked + +++Coffee +++ minus

Corn +++ minus

Cowrsquos milk ++ minus

Egg Yolk cooked ++ +++Gelatin ++ +++Hazelnut raw and roasted minus +Hemp +++ minus

Kamut +++ minus

Latex hevein +++ minus

Lemon and lime + +Macadamia nut raw and roasted ++ ++Millet ++ minus

Mustard seed +++ +++Oats +++ ++Orange Juice pasteurized or concentrate +++ minus

Peanut butter ++ ++Peanut roasted ++ +Pistachio raw and roasted + ++Potato +++ minus

Potato white baked + minus

Quinoa +++ minus

Radish + minus

Rice +++ minus

Rye ++ minus

Salmon raw + +++Scallops cooked +++ +++Seaweed +++ +++Sesame + minus

Shrimp cooked ++ +++Sorghum +++ minus

Soy Bean agglutinin + minus

Soy sauce gluten-free + +++Spelt ++Squid (calamari) cooked +++ +++Sunflower seeds roasted + minus

Tapioca +++ minus

Tofu +++ +++Tilapia cooked + +++

Table 2 Continued

Food protein T3

T4

Tuna cooked + +++Tuna raw ++ +++Yeast + minus

Zucchini cooked minus +++ = 066ndash086 ++ = 087ndash0107 +++ ge 108

of a 1 500 dilution of monoclonal anti-thyroid antibody andthe completion of all ELISA steps the OD were recorded

29 Inhibition of Anti-Thyroid Antibody Binding to FoodAntigen-Coated Plates by Different Concentrations of FoodAntigens Four different rows of three different microtiterplateswere coated in this patternwellsA1 B1 C1 andD1werecoated with T

3 the other eleven wells of each row (A2ndashA12

B2ndashB12 C2ndashC12 and D2ndashD12) were coated with roastedcashew egg seaweed or latex hevein Controls and inhibitorswere then added as follows 100 120583L of serum diluent to wellsA1-A2 B1-B2 C1-C2 andD1-D2 100 120583L of diluent containing100 120583g of T

3to wells A3 B3 C3 and D3 and 05ndash120 120583g

of cashew egg seaweed and latex hevein to respectivelyA4ndashA12 B4ndashB12 C4ndashC12 and D4ndashD12

Plates were incubated at 37∘C for 1 hour and 100 120583L ofmouse monoclonal anti-T

3was added to all 48 wells After

repeated incubation washing the addition of the secondaryantibody and completion of all the ELISA steps the opticaldensities were recorded Plate 2 was used for the addition ofT4and anti-T

4antibody Plate 3 was used for the addition of

Tg and anti-Tg with proper controls

3 Results

31 Immune Reactivity between Affinity-Purified PolyclonalAntibodies and Food Antigens The affinity-purified poly-clonal antibody made against TSH-R and monoclonal anti-body made against TPO did not react with any of 204 foodproteins and TBG But polyclonal antibody made againstDIO2 reacted only with buckwheat with a 1+ reaction

32 Reaction of Monoclonal Antibodies Made against Thy-roglobulin T3 and T4 with 204 Food Proteins Using thismonoclonal antibody against Tg resulted in a 3+ immunereaction with latex hevein (see Table 2)

Using monoclonal antibody against T4 our study found

a significant list of food proteins that directly demonstratedimmune reactivity with thyroxine These foods include avo-cado lemon and lime cooked Brussels sprouts seaweedcooked zucchini roasted and raw almond cooked black beanraw and roasted Brazil nut cashew roasted cashew cashewvicilin raw and roasted hazelnut raw and roastedmacadamianut mustard seed roasted peanut peanut butter raw androasted pistachio gluten-free soy sauce tofu gelatin cookedegg yolk raw salmon cooked tilapia raw tuna cookedtuna cooked clam cooked scallops cooked squid (calamari)cooked shrimp amaranth and oats (see Table 2)


Table 3 Example of 10 selected foods and their degrees of reactivitywith monoclonal antibodies against Tg T3 and T4

Tg T3

T4

Latex hevein +++ +++ minus

Kamut minus +++ minus

Soy sauce minus + +++Gelatin minus ++ +++Scallops minus +++ +++Cashew roasted minus ++ +++Cashew vicilin minus + +++Coffee protein minus +++ minus

Brazil nut minus + +++Almond minus minus ++

Similarly using monoclonal antibody made against T3

we found a significant list of food proteins that directlydemonstrated immune reactivity with triiodothyronineThese foods include avocado latex hevein lemon and limeorange juice (pasteurized and concentrate) cooked Brusselssprouts bakedwhite potato seaweed radish roasted almondraw and roasted Brazil nut cashew roasted cashew cashewvicilin raw and roasted macadamia nut mustard seedsroasted peanut peanut butter raw and roasted pistachiosoy bean agglutinin gluten-free soy sauce tofu roastedsunflower seeds gelatin cooked egg yolk raw salmoncooked tilapia raw tuna cooked tuna cooked clam cookedscallops cooked squid (calamari) cooked shrimp cowrsquosmilkcasein (alpha and beta) sesame hemp rye barley kamutbuckwheat sorghum millet spelt amaranth quinoa yeastoats corn and rice (see Table 2)

33 Simultaneous Reactivity of Monoclonal Antibodies againstThyroid Target Sites We selected 10 foods from Table 1 tosee if the same immune reactivity with a specific food forinstance latex hevein elicited by amonoclonal antibodymadeagainst T

3 for example would occur with a different mono-

clonal antibodyThe data clearly shows that each monoclonalantibody shows different patterns of immune reactivity to thefood antigens (see Table 3) In fact TPO and TBG had noreactions to any food at all

34 Demonstration of the Specificity of Anti-Thyroid Anti-bodies Binding to Different Food Antigens The specificity ofthese monoclonal anti-T

3 anti-T

4 and anti-Tg antibodies in

binding to various food antigens was confirmed by dilutionand inhibition studies As shown in Figures 1(a) 1(b) and 1(c)in proportion to the dilutions of the monoclonal antibodiesthe OD or immune reactions to the food antigens declinesignificantly For example the reaction of anti-T

3antibody

at a dilution of 1 500 with seaweed gives an OD of 28a dilution of 1 16000 gives an OD of 123 and a dilutionof 1 512000 gives an OD of 02 which is equivalent tothe background of the ELISA (see Figure 1(a)) Similarresults were obtained with the reaction of diluted T

4with

various food antigens (see Figure 1(b)) Interestingly using

anti-T4antibody we found significant differences between

the immune reactivity of raw cashew and roasted cashew(Figure 1(b))The binding of anti-Tg antibody to latex heveinbut not to seaweed egg or cashew and the proportion ofthe binding to antibody dilution are shown in Figure 1(c)Furthermore when fixed amounts of antibodiesmade againstT3 T4 and Tg were added to plates coated with food

concentrations of 0037ndash400120583gmL the ELISAOD increasedin proportion to the concentration of the food antigens(see Figures 2(a) 2(b) and 2(c)) The reaction of anti-T4with serially diluted roasted cashew was stronger than

with raw cashew (see Figure 2(b)) To further demonstratethe specificity of these antigen-antibody reactions differentamounts of food antigens (inhibitors) in concentrations of05ndash120120583g or controls were added in the liquid phase of platesthat contained the optimal concentrations of food antigensin solid phase The addition of anti-T3 anti-T4 or anti-Tgantibodies to the mixture resulted in a significant inhibitionof thyroid antibody binding to some food antigens on theplatesThis inhibition of antigen-antibody reaction was moreobvious when higher concentrations of food antigens wereused in the liquid phase (Figures 3(a) 3(b) and 3(c))

4 Discussion

Our laboratory study found dietary proteins that share aminoacid sequence homology and have the potential to play a rolein cross-reactivity with thyroid target sites This may serve tofill a knowledge gap and may have identified a starting placefor further research into the relationship between immuno-logical responses to dietary proteins and autoimmune thyroiddisease We identified immune reactivity between antibod-ies made against various thyroid target antigens and foodproteins and target sites on various locations of the thyroidaxisThis potential antibody against specific thyroid axis siteswith food antigens can lead to the possibilities that somedietary proteins may play a role in autoimmune thyroiddisease Our study identified T4 T3 Tg and 5-deiodinaseantibodies that reacted with purified food antigens Thesereactions suggest immunological cross-reactivity betweenfood immune reactions and thyroid axis sites

Interestingly although many pathogenic organisms haveshown direct cross-reactivity with both TSH-R and TPO ourstudy found that none of the 204 foods tested demonstratedany immune reactive response to these key autoimmunetarget sites associated with Gravesrsquo and Hashimotorsquos diseasewith the exception of latex hevein for thyroglobulinHevein isa lectin-like protein derived from Hevea brasiliensis (rubbertree) Although this lectin-like protein is not found in foodit is associated with the latex-fruit syndrome Approximately30ndash35 of individuals who are allergic to natural rubber latexshow cross-reactive IgE hypersensitivity to plant sources suchas avocado chestnut bell pepper kiwi peach and tomatoImmune cross-reactivity with latex or latex-fruit syndromemay potentially lead to Tg cross-reactivity in susceptibleindividuals who are reactive to natural rubber latex andassociated foods [28]


1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed

Roasted cashewCashew

Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)

Figure 1The binding of serially diluted T3 (a) T4 (b) and Tg (c)monoclonal antibodies to the same concentration of different food antigens


05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)

Figure 2 The binding of monoclonal antibody against T3 (a) T4 (b) and Tg (c) to serially diluted food antigens


0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food

B 120 60 30 15 8 4 2 1 05T3Concentration of inhibitors

and controls in g

Seaweed Egg

Latex heveinRoasted cashew

(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D

B 120 60 30 15 8 4 2 1 05Concentration of inhibitors

and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)

Figure 3 Inhibition of T3 (a) T4 (b) and Tg (c) antibodies with different concentrations of food antigens

Although direct food reactions did not occur with TSH-RandTPOmost commonly associatedwithGravesrsquo disease andHashimotorsquos there was significant cross-reactivity betweenboth T

4and T

3hormones found within the thyroid gland

with 25ndash35 food antigens (Table 2) [29 30]We also did not identify immune reactions to TBG in

our study suggesting food immune reactivity does not appearto have a role in thyroid hormone transport dysregulation

or on ratios of thyroid hormones bound to protein andtherefore foods should not impact ratios of bound comparedto unbound thyroid hormones or biomarkers such as T

3

uptakeOne reactionwas identified between Type II 5-deiodinase

(DIO2) thereby suggesting that food immune reactivity

with buckwheat may potentially impact T4to T3thyroid

conversion associated with nonthyroidal illness syndrome


Table 4 Reaction of monoclonal and polyclonal antibodies against 204 different foods

Antibodies Degree of reactionminus + ++ +++

Polyclonal antibody against TSH-R 204 0 0 0Polyclonal antibody against DIO

2203 1 0 0

Monoclonal antibody against TPO 204 0 0 0Polyclonal antibody against TBG 204 0 0 0Polyclonal antibody against Tg 203 0 0 1Polyclonal antibody against T3 151 18 16 19Polyclonal antibody against T4 172 7 7 18

(NTI) or euthyroid sick syndrome These findings suggesta potential new mechanism for NTI not reported in theliterature to the best of our knowledge

Most of the cross-reactive reactions occurred with T4

and T3 and many of the cross-reactive foods overlapped

with both hormones as T4and T

3have very similar amino

acid homology especially involving iodine sequencing [31]Some common patterns were identified with these reactionsincluding cross-reactions to many gluten-containing foodsand grains and foods that contain iodine structural sequencessimilar to thyroid hormones such as seaweed white potatonuts (almond cashew hazelnut and peanut) seafood andmore (Table 2)

There was also overlap between T4and T

3foods not asso-

ciated with iodine such as cooked Brussels sprouts lemonand lime radish tofu gluten-free soy sauce and cookedzucchini Some of these food proteins have been classified asgoitrogens however their impact on thyroid functionmay bedue to immune cross-reactivity instead of interference withiodine uptake as proposed in the goitrogenic model Thisis especially probable given that some of these goitrogenicfoods were tested in a modified cooked state to removegoitrogenic properties such as glucosinolates Immunologicalcross-reactivity with foods listed as goitrogenic may explainsome of the observed adverse reactions that occur in smallsubsets of thyroid patients although the mechanisms nowappear to be immunological rather than goitrogenic

Additionally there were many cross-reactive foods withT4and T

3that did not overlap with each other Although

T3and T

4have some structural similarity with each other

T4has two independent conformations in the crystal lattice

with significant differences in the outer phenyl ring geometrywhen compared to T

3 The major differences between T

4

and T3structures are shortened C41015840-O41015840 bond contraction

of the C31015840-C41015840-C51015840 angle and an increase in the C31015840 andcarbon C51015840 angles of T

4[31] These differences may explain

why certain food proteins only have cross-reactive reactionswith either T

3or T4 Overall this simultaneous reaction

of three different monoclonal antibodies made against TgT3 and T

4with some food antigens but not others is the

best indication for the specificity of the immune reactionsbetween antibodies made against thyroid target antigens anddifferent food proteins (see Tables 2 and 4)

The specificity of these anti-thyroid antibodies bindingto antigenic determinants of food antigens was shown by

serial dilution of food antigens or different concentrations ofmonoclonal antibody with the same concentration of foodantigens in the solid phase The dose response curves shownin Figures 1(a) 1(b) 2(a) and 2(b) with seaweed first followedby roasted cashew and cooked egg yolk and the curves inFigure 1(c) that show a strong response from latex heveinare supportive of the specificity of these antigen-antibodyreactions To further confirm this reaction of antibodieswith food antigens inhibition studies were conducted withdifferent concentrations of food antigens in the liquid phaseand the addition ofmonoclonal thyroid antibodies As shownin Figures 3(a) and 3(b) the addition of 120120583g of seaweed tothe liquid phase resulted in 76 inhibition in the binding ofanti-T

3and 80 in the binding of anti-T4 to the food antigens

(Figures 3(a) and 3(b)) The inhibition of Tg antibody toseaweed was not significant since to begin with the anti-Tg antibody did not react to seaweed (see Figure 3(c)) Incontrast the inhibition of anti-Tg binding to latex hevein wasvery significant (gt50) when 15ndash120 120583g of antigen was addedto the liquid phase Overall this inhibition of anti-thyroidantibody binding to different food antigens was reversed inproportion to the lower concentration of immune reactivefoods in the liquid phase The results of these experimentssupport the proposition that the binding of monoclonalantibodies to T3 T4 and Tg to different foods as shownin Tables 1ndash3 is specific and hence cross-reactivity betweenthyroid tissue antigens and various food antigens should betaken seriously

5 Conclusions

The results of our study identified immune reactivity betweenT3 T4 DIO

2 thyroglobulin and many food proteins These

immune reactions may explain the etiology of some casesof food interactions with both thyroid hormone replacementand thyroid hormone metabolism Theoretically these foodprotein reactions may contribute to autoimmune reactivityin a subset of autoimmune thyroid disease patients a subjectthat warrants further research

It is doubtful that the consumption of potentially reactivefood proteins alone would induce an inflammatory responseon thyroid axis target sites The consumption of dietaryproteins in combination with other factors such as digestiveenzymes is necessary to induce immunological reactions


First dietary proteins cannot have an impact on antigenic-antibody models if properly digested unless the individualis immunologically reactive to those foods and therebyproducing antibodies to those specific dietary proteins dueto a failure in oral tolerance to those specific undigested foodantigens [32] If the individual has oral tolerance to ingestedproteins and does not produce significant antibodies tothose proteins then no reaction would be expected Secondantigen-antibody reactions alone are not solely responsiblefor pathogenic reactions Cellular immunity immunologicaltolerance human leukocyte antigen (HLA) allele and otherfactors are involved with pathogenic immune reactions todietary proteins Therefore antibodies binding to antigensare not always pathogenic however the ability of specificmonoclonal and polyclonal antibodies to bind with purifiedproteins suggests the potential for immunological cross-reactivity in a subset of susceptible individuals

Further research should be conducted to evaluate thespecific epitope for each of these foods and thyroid axistarget sites Additionally immunological factors such astolerance T-cell polarization and other factors that mayinduce susceptibility to dietary protein immune reactivityneed to be investigated Individual case studies and clinicaltrials will be required to assess whether any actual clinical roleexists for these food proteins in thyroid-associated reactionsThe results of our research provide a list of susceptibledietary proteins that may immunologically impact thyroidinteractions and warrant further study These results providea first step in narrowing down a list of specific dietary proteinsthat due to protein cross-reactivity may potentially have animpact on autoimmune thyroid disease

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] L Tuft and G I Blumstein ldquoStudies in food allergy II Sensi-tization to fresh fruits clinical and experimental observationsrdquoJournal of Allergy vol 13 no 6 pp 574ndash582 1942

[2] R S Bonds T Midoro-Horiuti and R Goldblum ldquoA structuralbasis for food allergy the role of cross-reactivityrdquo CurrentOpinion in Allergy and Clinical Immunology vol 8 no 1 pp82ndash86 2008

[3] Y Saeki and K Ishihara ldquoInfection-immunity liaisonPathogen-driven autoimmune-mimicry (PDAIM)rdquo Autoim-munity Reviews vol 13 no 10 pp 1064ndash1069 2014

[4] R Root-Bernstein and D Fairweather ldquoComplexities in therelationship between infection and autoimmunityrdquo CurrentAllergy and Asthma Reports vol 14 no 1 article 407 2014

[5] A Marino Gammazza M Rizzo R Citarrella et al ldquoElevatedbloodHsp60 its structural similarities and cross-reactivity withthyroid molecules and its presence on the plasma membraneof oncocytes point to the chaperonin as an immunopathogenicfactor in Hashimotorsquos thyroiditisrdquo Cell Stress and Chaperonesvol 19 no 3 pp 343ndash353 2014

[6] C E Hargreaves M Grasso C S Hampe et al ldquoYersiniaenterocolitica provides the link between thyroid-stimulating

antibodies and their germline counterparts in Gravesrsquo diseaserdquoJournal of Immunology vol 190 no 11 pp 5373ndash5381 2013

[7] Z Wang Q Zhang J Lu et al ldquoIdentification of outermembrane porin F protein of Yersinia enterocolitica recognizedby antithyrotopin receptor antibodies in Gravesrsquo disease anddetermination of its epitope using mass spectrometry andbioinformatics toolsrdquo Journal of Clinical Endocrinology andMetabolism vol 95 no 8 pp 4012ndash4020 2010

[8] M Soveid K H Asl and G R Omrani ldquoInfection by cag apositive strains of helicobacter pylori is associated with autoim-mune thyroid disease in Iranian patientsrdquo Iranian Journal ofImmunology vol 9 no 1 pp 48ndash52 2012

[9] N Figura G Di Cairano F Lore et al ldquoThe infection byHelicobacter pylori strains expressing CagA is highly prevalentin women with autoimmune thyroid disordersrdquo Journal ofPhysiology and Pharmacology vol 50 no 5 pp 817ndash826 1999

[10] A Vojdani P Rahimian H Kalhor and E MordechaildquoImmunological cross reactivity between Candida albicans andhuman tissuerdquo Journal of Clinical amp Laboratory Immunologyvol 48 no 1 pp 1ndash15 1996

[11] R Tozzoli O Barzilai M Ram et al ldquoInfections and autoim-mune thyroid diseases parallel detection of antibodies againstpathogens with proteomic technologyrdquo Autoimmunity Reviewsvol 8 no 2 pp 112ndash115 2008

[12] E Gregoric J A Gregoric F Guarneri and S BenvengaldquoInjections of Clostridium botulinum neurotoxin A may causethyroid complications in predisposed persons based on molec-ular mimicry with thyroid autoantigensrdquo Endocrine vol 39 no1 pp 41ndash47 2011

[13] S Benvenga F Guarneri M Vaccaro L Santarpia and F Tri-marchi ldquoHomologies between proteins of Borrelia burgdorferiand thyroid autoantigensrdquoThyroid vol 14 no 11 pp 964ndash9662004

[14] M H S Kraemer E A Donadi M A Tambascia L A Magnaand L S Prigenzi ldquoRelationship between HLA antigens andinfectious agents in contributing towards the development ofGravesrsquo diseaserdquo Immunological Investigations vol 27 no 1-2pp 17ndash29 1998

[15] L H Duntas ldquoAutoimmunity does celiac disease triggerautoimmune thyroiditisrdquoNature Reviews Endocrinology vol 5no 4 pp 190-191 2009

[16] N R Lewis and G K T Holmes ldquoRisk of morbidity incontemporary celiac diseaserdquoExpert Review of Gastroenterologyand Hepatology vol 4 no 6 pp 767ndash780 2010

[17] A Vojdani ldquoDetection of IgE IgG IgA and IgM antibod-ies against raw and processed food antigensrdquo Nutrition andMetabolism vol 6 article no 22 2009

[18] A Bojarska-Szmygin and R Ciechanek ldquoAntibodies againstTSH receptors (TRAb) as indicators in prognosing the effec-tiveness of Tiamazol therapy for Graversquos Diseaserdquo Wiadomoscilekarskie (Warsaw Poland 1960) vol 56 no 7-8 pp 303ndash3072003

[19] M Rieu A Richard M Rosilio et al ldquoEffects of thyroidstatus on thyroid autoimmunity expression in euthyroid andhypothyroid patients with Hashimotorsquos thyroiditisrdquo ClinicalEndocrinology vol 40 no 4 pp 529ndash535 1994

[20] A Iervasi G Iervasi A Carpi and G C Zucchelli ldquoSerumthyroglobulin measurement clinical background and mainmethodological aspects with clinical impactrdquo Biomedicine andPharmacotherapy vol 60 no 8 pp 414ndash424 2006

[21] W M Wiersinga ldquoThyroid autoimmunityrdquo Endocrine Develop-ment vol 26 pp 139ndash157 2014


[22] A A Petruk M S Labanda R M S Alvarez and M A MartildquoThe allosteric modulation of thyroxine-binding globulin affin-ity is entropy drivenrdquo Biochimica et Biophysica Acta vol 1830no 6 pp 3570ndash3577 2013

[23] G Barbesino ldquoDrugs affecting thyroid functionrdquo Thyroid vol20 no 7 pp 763ndash770 2010

[24] R Arrojo E Drigo T L Fonseca J P S Werneck-De-Castroand A C Bianco ldquoRole of the type 2 iodothyronine deiodinase(D2) in the control of thyroid hormone signalingrdquo Biochimicaet Biophysica Acta vol 1830 no 7 pp 3956ndash3964 2013

[25] R Mullur Y-Y Liu and G A Brent ldquoThyroid hormoneregulation of metabolismrdquo Physiological Reviews vol 94 no 2pp 355ndash382 2014

[26] A Vojdani ldquoImmune reactivities to peanut proteins agglu-tinins and oleosinsrdquo Alternative Therapies in Health andMedicine vol 21 pp 73ndash79 2015

[27] A Vojdani and C Vojdani ldquoImmune reactivities against gumsrdquoAlternativeTherapies in Health andMedicine vol 21 pp 64ndash722015

[28] S Wagner and H Breiteneder ldquoThe latex-fruit syndromerdquoBiochemical Society Transactions vol 30 no 6 pp 935ndash9402002

[29] C Duthoit V Estienne A Giraud et al ldquoHydrogen peroxide-induced production of a 40 kda immunoreactive thyroglobulinfragment in human thyroid cells The onset of thyroid autoim-munityrdquo Biochemical Journal vol 360 no 3 pp 557ndash562 2001

[30] E Ademoglu N Ozbey Y Erbil et al ldquoDetermination ofoxidative stress in thyroid tissue and plasma of patients withGravesrsquo diseaserdquo European Journal of Internal Medicine vol 17no 8 pp 545ndash550 2006

[31] V Cody ldquoMolecular conformation of thyroid hormones Struc-ture and binding interactions of thyroxinerdquo Endocrine Researchvol 6 no 2 pp 123ndash134 1979

[32] A Vojdani ldquoOral tolerance and its relationship to foodimmunoreactivitiesrdquo Alternative Therapies in Health andMedicine vol 21 pp 23ndash32 2015

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Numerous gastrointestinal pathogens have demonstratedmolecular mimicry with thyroid tissue For example humanmonoclonal thyroid-stimulating hormone receptor (TSH-r)has been shown to cross-react with Yersinia enterocolitica(Y enterocolitica) thereby providing a mechanistic frame-work for molecular mimicry in Gravesrsquo disease where Yenterocolitica antibody production promotes cross-reactivepathogenic response to TSH receptor [6] Researchers havediscovered that the outer membrane of the porin F protein ofY enterocolitica shares cross-immunogenicity with a leucine-rich domain of TSH receptor and plays a role in inducingautoimmunity to TSH receptor through molecular mimicry[7]

Immune cross-reactivity between Helicobacter pylori (Hpylori) and autoimmune thyroid disease has been suspecteddue to correlations between early onset H pylori infectionsand Hashimotorsquos hypothyroidism and Gravesrsquo disease [8 9]Cross-reactivity with Candida albicans and thyroid antigenshas been identified and associated with the development ofautoimmune thyroid disease [10] The protozoa Toxoplasmagondii has also been reported to potentially induce autoim-mune thyroid cross-reactivity through molecular mimicrymechanisms [11]

In addition to gastrointestinal pathogens Clostridiumbotulinum neurotoxin A (Btx) has been reported to induceserum elevations of TSH Researchers found that Btx andthyroid autoantigens share amino acid sequence homologyandmay play a role in cross-reactive complication of autoim-mune thyroid disease [12] Borrelia burgdorferi has beenshown to have protein homology with TSH receptor andtherefore plays a role as an antigenic trigger for autoimmunethyroid disease [13] Coxsackie virus antibodies have alsodemonstrated cross-reactivity with the thyroid and have beenreported to be a contributing factor to the pathogenesis ofautoimmune thyroid disorder [14]

The list of pathogenic organisms such as viral bacterialfungal spirochete and protozoa antibodies that may con-tribute to tissue-specific thyroid autoimmunity via antibodyamino acid sequence homology is a growing field of studyHowever little research has been done with food proteinantibodies and their potential role in thyroid specific cross-reactivity Published research has looked at gluten and thyroidautoimmunity but the immunological reactive role that otherdietary proteins may play with thyroid function has been alimited area of research [15 16]

In this study we evaluated the potential for food cross-reactivity with the thyroid axis by evaluating immune reac-tivity between purified dietary proteins and thyroid targetspecific antibodiesWemeasured immune reactivity of eithertarget specific monoclonal or polyclonal antibodies for TSHreceptor 51015840deiodinase thyroid peroxidase thyroglobulinthyroxine-binding globulin thyroxine and triiodothyronineagainst 204 purified dietary proteins that are commonly con-sumed in raw or cooked forms Food determinants includedunmodified (raw) and modified (cooked and roasted) foodproteins herbs spices food gums brewed beverages andadditives We included raw versus modified food antigenssince in our earlier study we showed that some individualsmay react to raw food antigens but not heat-modified ones

while conversely others may react to heat-modified but notraw food antigens [17]

Specific target proteins along the thyroid axis associatedwith autoimmune thyroid disease were evaluated in our studyincluding thyroid-stimulating hormone receptor (TSH-R)51015840deiodinase thyroid peroxidase thyroglobulin thyroxine-binding globulin thyroxine and triiodothyronine TSH-Rantibody reactions are the hallmark of Gravesrsquo disease Theseantibody reactions promote autoimmune reactivity againstthe target protein but homosteric binding to these receptorscan promote increased production of thyroid hormonesleading to hyperthyroidism [18]Thyroid peroxidase (TPO) isthe key thyroid enzyme necessary for the synthesis of thyroidhormones and is the target for Hashimotorsquos autoimmunityTPO antibodies are associatedwith destruction of the thyroidand thyroiditis [19] Thyroglobulin (Tg) is a dimeric proteinsynthesized in the thyroid and used for production of thyroidhormones Its level is elevated with thyroid tissue breakdownsuch as with thyroiditis and differentiated thyroid cancer[20] Tg is a common target for thyroid autoimmunity [21]Thyroxine-binding globulin (TBG) is the transport proteinfor thyroid hormones in circulation and has high affinityfor thyroxine and triiodothyronine [22] Levels in the bodycan change due to metabolic disease pregnancy oral contra-ceptive use and hormone replacement therapy [23] Type IIiodothyronine deiodinase (DIO

2) is the enzyme that converts

prohormone thyroxine by outer ring deiodination to bioac-tive triiodothyronine [24] Thyroxine (T

4) is a prohormone

synthesized by the thyroid gland and composed of four iodinemolecules attached to thyroglobulin Triiodothyronine (T

3)

is bioactive thyroid hormone that is responsible for the keyphysiological mechanisms of thyroid target tissue function[25]

The identification of immunological food protein cross-reactivity in the laboratory with thyroid axis target sitesis the first step in understanding whether dietary proteinsmay potentially play an immunological reactive role inautoimmune thyroid disease In this study we attempt to takethe initial first step by determining any potential patternsof immunological cross-reactivity with a diverse list of foodproteins an specific thyroid axis autoimmune target sites

2 Materials and Methods

21 Polyclonal and Monoclonal Antibodies Affinity-purifiedrabbit polyclonal thyroid-stimulating hormone receptor anti-body affinity-purified goat polyclonal thyroxine 5-deiodinase(DIO2) antibody monoclonal antibody to thyroid perox-

idase monoclonal antibody against thyroglobulin mousemonoclonal thyroxine antibodymousemonoclonal antibodyto triiodothyronine and monoclonal antibody to thyroxine-binding globulin were purchased from MyBioSource Inc(San Diego CA USA)

22 Preparation of Dietary Antigens Food antigens wereprepared from products purchased from the supermarket inboth raw roasted or cooked form For that preparation 10 gof food product was put in a food processor using 01M ofphosphate buffer saline (PBS) at pH 74Themixer was turned















Table 1 Continued



Table 1 Continued


Table 1 Continued






Food protein T3

T4






Corn +++ minus



Kamut +++ minus






Quinoa +++ minus

Radish + minus

Rice +++ minus

Rye ++ minus





Tapioca +++ minus


Table 2 Continued

Food protein T3

T4














3 Results







Tg T3

T4











3 anti-T



3antibody


4with






4 Discussion




1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)



05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























Table 1 Continued



Table 1 Continued


Table 1 Continued






Food protein T3

T4






Corn +++ minus



Kamut +++ minus






Quinoa +++ minus

Radish + minus

Rice +++ minus

Rye ++ minus





Tapioca +++ minus


Table 2 Continued

Food protein T3

T4














3 Results







Tg T3

T4











3 anti-T



3antibody


4with






4 Discussion




1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)



05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued


Table 1 Continued






Food protein T3

T4






Corn +++ minus



Kamut +++ minus






Quinoa +++ minus

Radish + minus

Rice +++ minus

Rye ++ minus





Tapioca +++ minus


Table 2 Continued

Food protein T3

T4














3 Results







Tg T3

T4











3 anti-T



3antibody


4with






4 Discussion




1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)



05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Food protein T3

T4






Corn +++ minus



Kamut +++ minus






Quinoa +++ minus

Radish + minus

Rice +++ minus

Rye ++ minus





Tapioca +++ minus


Table 2 Continued

Food protein T3

T4














3 Results







Tg T3

T4











3 anti-T



3antibody


4with






4 Discussion




1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)



05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Tg T3

T4











3 anti-T



3antibody


4with






4 Discussion




1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)



05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 500

1 1000

1 2000

1 8000

1 4000

1 16000

1 32000

1 64000

1 128000

1 256000

Ant

ibod

y di

lutio

n

05 1 15 2 25 30ELISA OD

1 512000

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew

Cashew

Egg

(b)

05 1 15 2 25 30ELISA OD

1 500

1 1000

1 2000

1 4000

1 8000

1 16000

1 32000

1 64000

1 128000

1 256000

1 512000

Ant

ibod

y di

lutio

n

Seaweed

Roasted cashew Egg

Latex hevein

(c)



05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(a)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed


Egg

(b)

05 1 15 2 25 30ELISA OD

400

200

100

50

25

125

62

31

15

075

0037

Ant

igen

conc

entr

atio

n (

gm

L)

Seaweed Cashew

Egg Latex hevein

(c)



0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

05

1

15

2

25

3

35

4an

tigen

s exp

ress

ed as

OD

Ant

i-４3

bind

ing

to４3

and

food


and controls in g

Seaweed Egg


(a)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

DA

nti-４

4bi

ndin

g to

４4

and

food


and controls in g

Seaweed Egg


(b)

0

05

1

15

2

25

3

35

4

antig

ens e

xpre

ssed

as O

D


and controls in g

Tg

Ant

i-Tg

bind

ing

to T

g an

d fo

od

Seaweed Egg


(c)



4and T





3









2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2203 1 0 0









3foods not asso-




T3and T





4













5 Conclusions










References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















immunological reactivity using monoclonal and polyclonal...

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