Research Article Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame …downloads.hindawi.com/journals/bmri/2015/853836.pdf · 2019. 7. 31. · Immunogen Preparation

Research ArticleSandwich Enzyme-Linked Immunosorbent Assay forDetecting Sesame Seed in Foods

Stef J Koppelman1 Guumllsen Soumlylemez12 Lynn Niemann1 Ferdelie E Gaskin1

Joseph L Baumert1 and Steve L Taylor1

1Food Allergy Research and Resource Program Department of Food Science and Technology University of NebraskaLincoln NE 68588-6207 USA2The Ministry of Agriculture and Rural Affairs Directorate of Food Control and Central Food Research 16036 Bursa Turkey

Correspondence should be addressed to Stef J Koppelman stefkoppelmanzonnetnl and Steve L Taylor staylor2unledu

Received 29 September 2015 Revised 15 November 2015 Accepted 23 November 2015

Academic Editor Jose C Jimenez-Lopez

Copyright copy 2015 Stef J Koppelman 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

Small amounts of sesame can trigger allergic reactions in sesame-allergic patients Because sesame is a widely used food ingredientanalytical methods are needed to support quality control and food safety programs in the food industry In this study polyclonalantibodies against sesame seed proteins were raised and an enzyme-linked immunosorbent assay (ELISA) was developed forthe detection and quantification of sesame seed residue in food A comparison was made between this ELISA and other assaysparticularly focusing on recovery of sesame seed residue from different food matrices The developed ELISA is sensitive with alower limit of quantification of 05 ppm and shows essentially no cross-reactivity with other foods or food ingredients (92 tested)The ELISA has a good recovery for analyzing sesame-based tahini in peanut butter outperforming one other test In a baked breadmatrix the ELISA has a low recovery while two other assays perform better We conclude that a sensitive and specific ELISAcan be constructed based on polyclonal antibodies which is suitable for detection of small amounts of sesame seed relevant forhighly allergic patients Furthermore we conclude that different food products may require different assays to ensure adequatequantification of sesame

1 Introduction

Sesame (Sesamum indicum) is a seed crop native to Africathe Middle East and parts of Asia where it traditionallyforms an important part of the human diet Sesame is oneof the oldest cultivated crops owing to its nutritional valueand good resistance to draught Currently Myanmar is thelargest producer of sesame seeds followed by India andChina(FAOSTAT database from the Food and Agricultural Organi-zation of the United Nations) with an annual production ofapproximately 70000 tons compared to the US productionof approximately 2500 tons annually Traditionally sesameseeds are consumed as a paste (tahini and a sweetened formcalled halvah) that serves as ingredient for many dishes Alsoin particular in the Western countries sesame seeds are usedfor toppings on bakery products like bagels hamburger bunsand crackers Sesame oil is also popular due to its unique

flavour profile The recent focus on health foods vegetarianfoods and ethnic foods in the Western countries has ledto an increase in the consumption of sesame seeds andsesame-derived products with the black and white sesamebeing the most commonly consumed varieties In parallelconcerns have emerged about the allergenicity of sesameseeds Compared to allergies for milk and egg the prevalenceof sesame allergy is low and varies regionally [1] In USAthe prevalence of sesame allergy is estimated at 01 basedon a nationwide cross-sectional random telephone surveythat included a total of 13534 subjects from 5300 households[2] Sesame seed allergy is more common in Israel [3] wheresesame seed accounts for a large part of the severe IgE-mediated reactions to food [4] and represents the thirdleading cause of food allergy after milk and egg Differencesbetween countries in the prevalence of sesame seed allergy arelikely due to dietary habits In the Middle East sesame seeds

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 853836 10 pageshttpdxdoiorg1011552015853836

2 BioMed Research International

are introduced in the diet at an early age as a good source ofcalories and bioavailable iron Thus sensitization to sesameseed and clinical sesame seed allergy before age of 2 years isdocumented [4]

Allergic reactions to the ingestion of sesame seed can betriggered by doses as low as 1mg of sesame seed protein forindividual cases and the population threshold study showedthat approximately 5mg of sesame seed protein triggers areaction in 10 of the sesame seed-allergic population [5]Sesame seeds contain several allergenic proteins that havebeen identified and partially characterized [6ndash9] Sesame oilcan also present a hazard to sesame seed-allergic individualsas it is often not highly refined and thus contains proteinresidues [10]

Unintentional ingestion of sesame seed is a risk forsesame seed-allergic consumers The presence of sesameseeds and ingredients derived from sesame seeds is not alwaysobvious Sesame seed flour paste and oil are not easilyidentified if products are not appropriately labelledThe seedsare small and a few seeds can easily be indistinguishable in abakery formulation Sesame seed residues can also occur infoods from cross-contact through use of shared processingequipment and facilities A need exists for analytical methodsthat are able to sensitively and specifically detect residuesof sesame seed Immunoassays for the detection of sesameseed proteins [11 12] and PCR methods for the detection ofDNA fragments [13 14] from sesame seed have previouslybeen described and commercial immunoassay kits exist forthe detection and quantification of sesame seed residuesPreliminary data from our group indicated that the variousmatrices in which sesame seed can potentially be presentcomplicated the quantification of sesame seed residues infood products While some methods seem appropriate forbakery products others may be more suitable for tahini Theaim of this study is to develop an ELISA for the detectionand quantification of sesame seed residues in foods andto compare its performance with regard to sensitivity andrecovery from various food matrices with two commerciallyavailable immunochemical assays for sesame seed

2 Materials and Methods

21 Immunogen Preparation Black (India black) and white(Mexico white hulled) sesame seeds were purchased fromPenzeys Ltd (Penzeys Spices Madison WI) and stored atroom temperature until needed Sesame seeds were washedthoroughly 1 8 (wv) with distilled water 6 times and driedunder a fume hood After drying the seeds were groundto uniform consistency using a blender (Oster ProfessionalProducts Chicago IL) Ground white and black sesameseeds were mixed and defatted by washing with ethyl ether(1 5 wv) with continuous stirring for 5 minutes the processwas repeated 5 timesThedefatted residuewas then allowed tosettle for 30 minutes and after that time the supernatant wasdiscarded At the last washing step the mixture was collectedusing a Buchner funnel fitted with Whatman number 1 filterpaper (Whatman International Ltd Maidstone England)and a final washing of the defatted sesame seeds was donetwice with acetone (1 5 wv) The defatted material was then

dried under a fume hood at room temperature for 48 hoursThis material was used for the immunization of animals Thecrude protein content of the material was determined usingthe Kjeldahl method Immunogen was prepared by emulsify-ing the dried material with adjuvant rather than first makingan extract in order to target the largest possible diversity ofepitopes including those from lipophilic proteins CompleteFreundrsquos adjuvant (CFADifco LabsDetroitMI)was used forpreparting the primary immunogen and incomplete Freundrsquosadjuvant (IFA) (Difco Labs Detroit MI) was used to prepareimmunogen for booster immunizations

22 Polyclonal Antibody Production One goat was immu-nized with 10mg of defatted sesame seed immunogenemulsifiedwithCFAand administrated subcutaneously (sc)The first booster injection comprised of 500120583g sesame seedimmunogen in IFA was administered 2 weeks after the initialimmunization Subsequent booster injections were admin-istered with 250120583g of defatted sesame seed in IFA every 3weeks after that alternating between sc and intramuscularly(im) The first test bleed was taken after the second boosterinjection (approximately 5 weeks after the initial injection)and thereafter test bleedswere taken 10 days after each boosterinjection

Three chickens were initially immunized with defattedsesame seed immunogen in CFA followed by booster injec-tions at 2-week intervals using 200120583g in IFA (sc and imalternatively) basically according to Schade et al [15] Eightweeks after the initial immunization this group was restedfor approximately 6 weeks and then booster injections werechanged to 3-week intervals due to an observed decrease inantibody production

Titer development was monitored using a noncompet-itive ELISA basically as described by Hefle et al [16] Inshort microtiter plates (MaxiSorp Nagle Nunc InternationalRoskilde Denmark) were coated with 1 120583g sesame seedproteinmL of coating buffer (0015M Na

2CO3 0035M

NaHCO3 and 002NaN

3 pH 96) and incubated overnight

at 4∘C After blocking with 350 120583L of PBS containing 01gelatin (porcine 300 bloom Sigma Chemical Co St LouisMO) sera from goat or chicken egg yolks were incubatedin various dilutions Plates were washed with PBS con-taining 005 Tween 20 and bound goat IgG and eggIgY were detected and quantified using commercial anti-immunoglobulin-alkaline phosphatase conjugates (anti-goatIgG [Pierce Chemical Co Rockford IL] anti-chicken IgY[Promega Co Madison WI]) respectively following themanufacturerrsquos instructions Bleeds from the goat with titersabove 10000 were pooled Yolks with titers gt3000 werepooled

23 Antibody Isolation Polyclonal IgG antibodies were par-tially purified from the sera by precipitation in 50 and35 ammonium sulfate [16] The partially purified IgG wasbriefly dialyzed against deionized water and then extensivelydialyzed against 001M phosphate-buffered saline (PBS)(0002M NaH

2PO4 0008M Na

2HPO4 085 NaCl and

002 NaN3 pH 74) as described by Hefle et al [16] This

IgG fraction was used as the coating antibody for ELISA

BioMed Research International 3

development Immune eggs were collected and stored at 4∘Cuntil needed The IgY fraction was purified using a com-mercial EGGstract IgY purification system (Promega CoMadison WI) Immunoglobulin Y preparations were storedatminus20∘Cuntil neededThis IgY fractionwas used as detectionantibody in the ELISA development The protein contentof the IgG (485mgmL) and IgY (77mgmL) fractions wasdetermined by the Lowry method [17]

24 Gel Electrophoresis and Immunoblotting Thereactivity ofthe polyclonal chicken and goat antibodies was characterizedby means of sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and immunoblotting Samplesof white sesame and black sesame extract (10 120583gwell) wereseparated under reducing conditions using the Laemmli[18] method with minor modifications Electrophoresis wasperformed using a Bio-Rad Mini Protean II (Bio-Rad Lab-oratories) with Ready Gel precast gels (15 Tri-HCl Bio-Rad Laboratories) Proteins were located on the gels bystainingwith brilliant blueG-colloidal stain and then scannedwith the Kodak Gel Logic 440 imaging system (KodakScientific Imaging Systems New Haven CT USA) Proteinsfrom unstained SDS-PAGE gels were transferred to polyvinyldifluoride (PVDF) membranes (Immobilon-P PVDF mem-brane 045120583m Millipore Corporation Billerica MA USA)as described by Towbin et al [19] The membranes were cutsuch that part of the lanes could be stained for protein usingIndia ink The remaining part of the membranes was usedfor immunoblotting and was blocked with 02 BSA in PBSpH 74 Chicken egg yolk (IgY) and goat (IgG) antibodies tosesame seed were tested in 5000- and 10000-fold dilutionsas primary antibodies using PBS-T (005 Tween 20) con-taining 02 BSA RIA grade (USB Corporation ClevelandOH USA) Then the membrane was incubated with eithercommercial anti-chicken IgY or anti-goat IgG peroxidaseconjugates (Promega Co MadisonWI and Pierce ChemicalCo Rockford IL resp) The membranes were washedthree times with PBS containing 005 Tween 20 (PBS-T)and then treated for at least 15 minutes with DAB (310158403-diaminobenzidine) substrate solution (Pierce BiotechnologyInc) prepared according to the manufacturerrsquos recommen-dation After washing with PBS-T the membranes were air-dried overnight at room temperature Digital images of thestained dried PDVF membranes from both protein stainingand immunoblotting experiments were then captured usinga Kodak Gel Logic 440 imaging system and analyzed withthe Kodak Gel Logic ID v365 software (Kodak ScientificImaging Systems)

25 Sandwich ELISA The general approach for the devel-opment of the sandwich ELISA was as follows Optimalconcentrations of coating antibody and detection antibodywere determined by varying the concentration of the coat-ing antibody in one dimension of microtiter plates andthe concentration of the detecting antibody in the otherdimension using various antigen concentrations and therelevant controls Antibody concentrations were chosen suchthat the sensitivity of the ELISA was sufficient while thenonspecific binding (background) was still low This led to

the following ELISA protocol Microtiter plates (MaxiSorpNagle Nunc International Roskilde Denmark) were coatedwith 100 120583L of goat anti-sesame seed antibody at 1 120583gmLin coating buffer (0015M Na

2CO3 0035M NaHCO

3 and

002 NaN3 pH 96) and incubated overnight at 4∘C All

subsequent incubations were carried out at 37∘C for 1 h(except incubation for color development with substrate)Plates were washed with PBS containing 005 Tween 20and 002 NaN

3(PBS-T) after each incubation step using a

programmable automatic platewasher (Dynatec LaboratoriesInc Chantilly VA) Plates were blocked with 350 120583L ofPBS containing 01 gelatin (porcine 300 bloom SigmaChemical Co St Louis MO) per well for one hour Samplesand standards were diluted in incubation buffer (PBS buffercontaining 01 bovine serum albumin RIA grade SigmaChemical Co St LouisMO) and applied towells in triplicateon each plate with a one-hour incubation After washingthree times 100 120583L of anti-sesame seed chicken egg yolk IgYantibody at 175 120583gmL in incubation buffer was then addedand incubated for one hour followed by washing four timesThen 100 120583L of commercial goat anti-chicken IgY-alkalinephosphatase conjugate (Promega Madison WI) was addedto each well diluted 2000-fold in incubation buffer followedby a one-hour incubation After washing four times 100120583L ofp-nitrophenyl phosphate (SigmaFast Sigma St Louis MO)was added and incubated for 30min at room temperaturefor color development Color development was stopped byaddition of 100 120583L of 01 N NaOH The absorbance wasmeasured at 410 nm using a MR5000 plate reader (DynatechLaboratories Inc Chantilly VA)

26 Standard Curve for Sandwich ELISA Standard curveswere constructed with extracts of bread mix spiked withknown amounts of sesame in the form of sesame seed flourThereto 454 grams of dry bread mix (Hodgson Mills lotnumber 10 07 09 2) was mixed with required amount ofsesame seed flour to achieve 1000 ppm in the final recipeThis amount will result in a concentration of 001 or100 ppm once the other ingredients are added (total weightof 742 g) Another 454 grams of dry bread mix served asnegative control To 454 grams of dry bread mix 30 grams ofvegetable oil 6 grams of fresh yeast and 252 grams of waterwere added resulting in a total weight of 742 grams Extractswere made of the 1000 ppm spiked bread mix and 0 ppmspiked breadmix (as described below) and these extractsweremixed in different ratios to obtain the dilutions required forthe standard curve Standard curves were generated usingPrism graphics software (GraphPad Prism Inc San DiegoCA)

27 Comparison with Commercial Sesame Seed Assays Com-mercially available ELISA kits for sesame seed were obtainedfrom Tepnel Biokits (Deeside United Kingdom) and ELISASystems (Windsor Australia) Assays were provided as readyto use kits with all reagents diluents and standards includedand were performed according to themanufacturerrsquos instruc-tions The Tepnel Biokits assay had a claimed range ofdetection of 6 to 100 ppm the ELISA Systems assay of 05 to


5 ppm Extracts were prepared according to the instructionsof the kit manufacturers

28 Food Samples All food samples were purchased fromlocal retailers in Lincoln Nebraska Ninety-seven foods orfood ingredients were evaluated for cross-reactivity andmatrix effects in the ELISA All samples were ground to uni-form consistency using an Oster blender (Oster ProfessionalProducts Chicago IL) or a mortar and pestle depending onthe texture of the samples

29 Naturally Incurred Food Bread Spiked with Sesame SeedFlour Bread mix spiked with 100 ppm sesame seed (assesame flour) and negative control bread mix were preparedas described for the preparation of the standard curve ofthe sandwich ELISA The spiked bread mix and the negativecontrol bread mix were mixed in different ratios such thatconcentrations of 100 ppm 50 ppm 10 ppm 5 ppm 1 ppmand 0 ppmwere reached in the final bread From these mixesbread was baked using an automatic bread baker (defaultsettings) After cooling to room temperature samples weretaken from different locations from the bread

210 Naturally Incurred Food Peanut Butter Spiked withTahini Tahini and peanut butter were obtained from a localsupermarket A spiked standard of 10000 ppm sesame seed inpeanut butter was prepared by mixing tahini and peanut but-ter in a 1100 (WW) ratio The mix was blended extensivelyusing a kitchen blender The 10000 ppm product was used tospike peanut butter in the following concentrations 100 ppm50 ppm 10 ppm 5 ppm 1 ppm and 0 ppm All samples wereblended extensively before analysis

211 Preparation of Extracts for ELISA Samples seeds wereground with a coffee grinder before extracting Preliminarydata indicated that less rigorousmethods of grinding gave rel-atively low recoveries when intact sesame seeds were present(not shown) Ground samples were extracted 1 10 (wv) in001M PBS for 2 h at room temperature with gentle shakingusing a Labquake shaker (BarnsteadThermoline DubuqueIA) Extracts were clarified by centrifugation at 3200timesg at10∘C in a Sorvall Centrifuge (Kendro Laboratory ProductsNewtown CT) Fresh extracts were used and stored at 4∘Cfor up to 5 days

Extracts for cross-reactivity testing were tested in undi-luted form 10-fold diluted from and 100-fold diluted formand results were calculated as ppm sesame seed equivalenceusing 1000000 ppm reactivity for sesame seed by definition

3 Results and Discussion

31 Characterization of Antibodies Crude extracts of whiteand black sesame seed were used for developing polyclonalantibodies for a sandwich ELISA to detect and quantifysesame seed residues in foods The advantage of polyclonalantibodies compared to monoclonal antibodies is that poly-clonal antibodies are more robust in detecting antigens infood products that have been exposed to food processing

A monoclonal antibody may become less reactive if its epi-tope is affected by food processing For polyclonal antibodiesmore epitopes play a role and overall reactivity will onlypartly be affected when a certain epitope is affected by foodprocessing Furthermore because we choose to work withwhole extracts rather than purified or isolated proteins thenumber of epitopes is even larger diminishing the chance oflosing reactivity in the ELISAwhen the reactivity of one of theepitopes is affected by food processing The goat polyclonalIgG antibody that was used for coating recognizes a broadrange of sesame seed proteins both for black and whitesesame seed The polyclonal IgY antibody that was used asthe detection antibody was raised in chickens and obtainedfrom egg yolk This polyclonal antibody also recognized abroad range of proteins both in black and white sesame seeds(Figure 1) Both polyclonal antibodies show good reactivityfor all major protein bands present in both extracts Proteinsin black sesame seed extract at approximately 20 and 60 kDawere comparably somewhat less reactive Several sesame seedallergens have been identified in the past decades An earlystudy reported that proteins in the range of 14ndash25 kDa wereimportant IgE-binding proteins for a group of 12 Frenchpatients [6] In a study with 20 sesame seed-allergic patientsfromUSA Beyer et al [7] found IgE binding towards sesameseed proteins in the range from 7 to 78 kDaThe proteins withthe lower molecular weight were assigned as Ses i 1 and Ses i2 and appear to be 2S albumins known as potent allergensfrom other seeds nuts and legumes as well Beyer et al [7]found that 75 of their patients had IgE-reactivity to the45 kDa 7S albumin a protein with a high sequence homologywith the important peanut allergen Ara h 1 This allergenwas assigned Ses i 3 Leduc et al [8] identified two otherpotentially important sesame seed allergens as oleosins in the15ndash17 kDa range in a French population of 32 sesame seed-allergic patients these two oleosins were designated as Ses i 4and Ses i 5 Work on serology of an Italian patient population(119899 = 18) indicated an important role for the 11S globulin Sesi 6 and Ses i 7 in particular its basic subunit of approximately25 kDa [9] Although we did not identify the proteins andallergens that our polyclonal antibodies were reactive to wesee good reactivity in the lowmolecular weight region typicalfor 2S albumins and oleosins probably reflecting reactivityto Ses i 1 Ses i 2 Ses i 4 Ses i 5 andor the basic subunitof Ses i 6 and Ses i 7 Furthermore we see clear reactivityof both polyclonal antibodies at the 45 kDa region probablycorresponding to Ses i 3 Because sesame seed is used as afood ingredient as the intact seed as flour or as a paste madefrom the whole seed it is unlikely that individual allergensbecome separated from each other during food processingTherefore if a set of antibodies recognizes several proteins orallergens they are potentially suitable for use in developmentof an ELISA to detect sesame seed residues in a range of foodproducts

32 Sensitivity of the ELISA With the polyclonal antibodiesraised in goat and chicken a sandwich ELISA was developedusing the goat antibody as coat and the chicken egg yolk IgYantibody as the detector The sandwich ELISA was tested forsensitivity by measuring a range of known concentrations


MW

Black sesameWhite sesame

SDS-PAGE Immunoblot blotSDS-PAGE Immunoblot blot

WS BS

200

21

14

65

45

97

37

IgG 5kIgY 5k IgG 10kIgY 10k IgG 5kIgY 5k IgG 10kIgY 10k

Figure 1 SDS-PAGE and immunoblot of sesame MW molecular weight markers (indicated left in kDa) WS white sesame extract and BSblack sesame extract Blots developed with egg yolk (IgY) or sheep (IgG) antibodies diluted 5000-fold (5 k) or 10000-fold (10 k)

Abso

rban

ce at

410

nm

00

05

10

15

20

25

01 1 10 100 1000001ppm sesame flour in bread mix

Figure 2 Calibration curve of sesame ELISA Data points are meanof nine independent experiments error bars represent the standarddeviation

of sesame seed flour extract spiked into a bread mix InFigure 2 a typical example of the average of 9 independentsassays is shown The sensitive range is from 015 to 37 ppmof sesame seed in bread mix The lowest tested standard was002 ppm however the response of this sample was close tothe background signal Samples of 05 ppm consistently gaveabsorbances that were well above the background signal andwe therefore used 05 ppm as lower limit of quantification

(LLOQ) Interpolation between 05 and 002 ppm can bedone but the calculated results may be imprecise

Limited information exists regarding the minimum doseof sesame seed proteins needed to elicit an allergic reactionDouble-blind placebo controlled food challenge (DBPCFC)threshold studies with large numbers of patients are availablefor some allergenic foods such as peanut [20ndash22] The pop-ulation threshold for sesame seeds can also be estimated butless precisely due to the lack of data from sesame seed-allergicindividuals [5 22]TheAllergen Bureau of Australia andNewZealand has established a Reference Dose for sesame seed of02mg sesame seed protein as a part of their VITAL programbased upon statistical dose distributionmodelling of availablepublished data on individual sesame seed thresholds [22] Aswith peanut [20 22] considerable variability seems to existregarding individual threshold doses among sesame seed-allergic patients There are a few studies that report levelsof sesame seeds that caused reactions One study based on9 sesame-allergic patients from France showed that allergicreactions were triggered with 100mg sesame seed flour [23]while other patients also from France reacted at substantiallyhigher dosages of 10 grams [6]The lowest reported thresholdfor sesame protein was 1mg [5] corresponding to 2mg ofseed flour In a population of 35 individuals 5mg of sesameprotein (corresponding to 10mg sesame flour) triggered areaction in 10 of the sesame-allergic patients [5] Casereports also appear to indicate that small amounts of sesameseed [24] or sesame-based tahini [25] can elicit severe allergic


reactions but the exact amount ingested was not known Inone of the cases the threshold dose for the individual wasdetermined afterwards and appeared to be 2 grams [24] Thestatistical models used in the VITAL program are inherentlyconservative and no evidence exists of sesame seed-allergicpatients reacting to 02mg of sesame seed protein (067mgsesame seed flour based upon 30 protein in sesame flouror 12mg sesame seed based on 17 protein in sesame seed)[5 22] Using the conservative lower threshold of 1mg sesameseed and assuming an intake of 100 grams per portion arelevant concentration of sesame residue that may trigger anallergic reaction is 10 ppmThe LLOQ of our assay (05 ppm)is well below this value and therefore suitable to detectsesame seed residue present in foods at clinically relevantconcentrations

Other assays to detect sesame seed residues in foodproducts have been described previously based on eitherimmunochemical methodology or on DNA-amplificationmethodology The sensitivity of the immunochemical meth-ods varied from 06 ppm sesame protein (sandwich ELISA[12]) to 5 ppm sesame protein (inhibition ELISA [11]) Thesensitivity of the DNA-amplification method was in onereport 50 ppm [13] and 5 ppm in another report [14] Atleast one of these methods by Schoringhumer et al [13] maynot detect clinically relevant levels of sesame seed residuesbased upon the VITAL Reference Dose [22] Furthermorethe DNA-amplification methods detect DNA rather thanthe allergenic proteinaceous part of sesame seeds Resultsobtained with DNA-amplification methods should thereforebe interpreted with more care especially for processed foodproducts containing sesame seed-based ingredients wherethe allergenic protein part is separated from the DNARNAfraction

33 Specificity of the ELISA The specificity of the sandwichELISA was investigated by testing extracts of 92 food ingre-dients in serial dilutions Table 1 shows the cross-reactivityof these food ingredients expressed as ppm equivalence ofsesame seed flour in which the reactivity of sesame itself isdefined as 1000000 ppm sesame seed flour The majority ofthe evaluated samples were negative at all tested dilutionsAbout a quarter of the samples were somewhat responsivein their undiluted form and only a few were still reactiveat a 10-fold dilution For the 100-fold dilutions only kidneybeans and allspice were just above two times the backgroundresulting in a cross-reactivity of 33 and 20 ppm sesameseed flour respectively Compared to sesame seed this isstill very low more than 100000-fold less than sesame seedWhile the specificity of the DNA-based methods for allergendetection is often very high as was the case for sesame [13 14]immunoassays based on polyclonal antibodies are sometimesnot very specific The indirect competitive ELISA using asingle polyclonal antibody was developed by Husain et al[11] which showed substantial cross-reactivity of 07 (or7000 ppm sesame seed protein) for one of the 13 tested foodingredients The combination of different independentlyraised polyclonal antibodies into a sandwich ELISA as wasdone in our study can at least partially overcome this issue[12] The high specificity of our method allows applying the

Table 1 Cross-reactivity of food ingredients in the sesame sandwichELISA

Ingredient description Cross-reactivity (ppm)Almonds (raw) lt1Anise seed 101Apple (fresh) 112Apple (dried) lt1Apricot (dried) lt1Banana (fresh) lt1Banana (dried) lt1Bell peppers (green fresh) lt1Bell peppers (red fresh) lt1Bell peppers (red roasted) lt1Brazil nuts lt1Cacao lt1Canola oil lt1Caraway seed 122Cashew lt1Celery seed 155Cherries (sweet pitted) 126Cherries (tart red pitted) 102Cheese (Parmesan) lt1Cheese (Romano) lt1Chick peas (fresh) lt1Chick peas (dried) lt1Chocolate (dark) lt1Chocolate (milk) lt1Cinnamon 189Cloves lt1Coconut lt1Corn meal lt1Corn starch lt1Corn syrup (dark) lt1Corn syrup (light) lt1Cumin 159Curry powder 149Dates (dried pitted) 118Fennel seed 163Garlic (fresh) lt1Garlic powder 123Hazelnut (roasted) lt1Honey lt1Kidney beans 334Kiwi (fresh) lt1Lemon (fresh) lt1Macadamias (roasted) lt1Mace 184Mango (dried sweetened) lt1Marjoram lt1Milk (1 low fat milk) lt1Molasses (unsulphured) 118


Table 1 Continued

Ingredient description Cross-reactivity (ppm)Mushroom (portobello) lt1Mushroom (shiitake) lt1Mustards seed (yellow) lt1Nutmeg 183Oat (whole grain) lt1Onion (fresh) lt1Onion (dried powder) lt1Oregano lt1Paprika 167Parsley flakes (dried) lt1Peaches (fresh) 101Peanut (roasted) lt1Peas (green dried) lt1Pecan (fresh) 103Pecan (roasted) lt1Pepper (black) 145Pine nuts lt1Pistachios (dry roasted) lt1Raisins lt1Raspberries 130Rice (dried) lt1Rye (flour whole grain) lt1Salt lt1Semolina lt1Sesame oil (extra virgin) lt1Soy flour lt1Soy milk lt1Soy nuts (roasted salted) lt1Soy oil lt1Strawberry (fresh) lt1Sugar (white) lt1Sugar (light brown) lt1Sunflower seeds (salted) lt1Thyme (dried) lt1Tomato paste lt1Tomato puree lt1Vanilla extract lt1Walnuts (black) lt1Wheat flour lt1Yeast (active dried) lt1Mix bread flour lt1Mix cake flour lt1Mix spices (unspecified) 205

method for a wide variety of food products Interestinglysesame oil did not react in our ELISA presumably becausethe oil was sufficiently refined and did not contain detectableamounts of (sesame) protein Similar observations of absenceof allergen in oils were also made for peanut oil [26] andmustard oil [27]

34 Comparing Different Immunoassays for Sesame SeedRecovery from Different Matrices Food processing con-ditions in particular heat treatment like baking oftenresult in decreased recovery of allergens when assayed withimmunoassays Also food products that have a high-fatcontent are known to be difficult matrices for detection ofallergen residues [28] For sesame seed the recovery fromwhole grain bread was described to be lower than expected[11] We compared our currently developed assay with twocommercially available assays for detection of sesame seedresidue with regard to recovery from difficult food matricesTwo model food products were prepared bread baked fromflour spiked with sesame seed flour and peanut butterspiked with tahini For the commercial assays the extractionprocedure recommended by the kit manufacturer was used

Model breads were baked with known amounts of sesameseed flour spiked into the bread mix before preparing thedough After baking samples were taken from different loca-tions in the bread (middle part lighter crust end and darkercrust end) Table 2 shows the recovery from the differentsamples for the three tested assays Some test results werebelow the LLOQ of the respective assays These values wereestimated by analyzing diluted standards and interpolationbetween the lowest tested standard and negative controlThisapproach is not truly quantitative but because it gives anindication of the recovery of sesame seed in food productsspiked with a low level of sesame seed these values wereincluded in Table 2 There are clear differences between theassays The current method and the kit from ELISA Systemsshow poor recoveries The mean recoveries for the currentmethod and the ELISA Systems kit were 65 and 13respectively Neither of these two assays could detect thelowest spike concentration of 1 ppm sesame flourTheTepnel-Neogen kit showed a better recovery with a mean of 39It was expected that the middle part of the bread wouldshow the highest recovery because this was less intensivelyexposed to heat while the darker crust end would show thelowest recovery However there was no correlation betweenexposure to heat and recovery (Table 2)

Our sesame seed ELISA was also compared to the twocommercial ELISA kits after spiking peanut butter withknown amounts of tahini (Table 3) As explained for Table 2diluted standards were again used to obtain informationon the recovery of sesame seed residue in food productsspiked with a low level of sesame seed paste Again this isnot a true quantitation but data are included in Table 3 forinformational purposes For the tahini model food productthe current assay showed the best recovery (mean 83)while the Tepnel-Neogen assay led to an overestimation ofthe sesame content (mean recovery 239) The assay fromELISA Systems had a mean recovery of 6 and could notdetect the lowest spike concentration of 1 ppm seed paste

Recovery of food allergens from food products for detec-tion and quantitation has been topic of investigation for quitesome time The majority of the published work is on peanutdetection Recovery from chocolate products and cookieshas been tested for different (commercially available) assaysin ring trials One particular study that included 5 differentassays for peanut residues and 30 European laboratories


Table 2 Recovery of sesame form bread spiked with sesame flour using different ELISA methods

Bread sample Spike level Current method1 Neogen-Tepnel2 ELISA Systems3

Sesame (ppm) Recovery () Sesame (ppm) Recovery () Sesame (ppm) Recovery ()

Middle part

0 mdash na mdash na mdash na1 mdash na 034 34 016 165 043 9 145 29 085 1710 061 6 456 46 115 1250 228 5 243 49 65 13100 653 7 218 22 144 14

Lighter crust end

0 BLQ na mdash na mdash na1 018 18 095 95 018 185 049 10 26 52 063 1310 049 5 43 43 118 1250 297 6 175 35 573 11100 729 7 423 42 133 13

Darker crust end


1 defined LLOQ is 05 ppm Values between 01 and 05 ppm may be therefore imprecise2 LLOQ given by manufacturer is 6 ppm Values below 6 ppm may be therefore imprecise3 LLOQ given by manufacturer is 05 ppm Values below 05 ppm may be therefore imprecisena not applicable

Table 3 Recovery of sesame form peanut butter spiked with tahini using different ELISA methods

Spike level Current method Neogen-Tepnel1 ELISA Systems2

Sesame (ppm) Recovery () Sesame (ppm) Recovery () Sesame (ppm) Recovery ()0 mdash na mdash na mdash na1 071 71 253 253 009 95 384 77 124 248 032 610 751 75 241 241 053 550 432 87 119 238 209 4100 104 104 214 214 411 41 LLOQ given by manufacturer is 6 ppm Values below 6 ppm may be therefore imprecise2 LLOQ given by manufacturer is 05 ppm Values below 05 ppm may be therefore imprecisena not applicable

found recoveries ranging from 44 to 191 for low ppm spikes[29] For sesame seed assays there are no such comprehensivering trials Of the two reported immunoassays to detectsesame seed residues the recoveries ranged from 70 to 160[11] and 42 to 145 [12] No other assays were used in thesestudies and only bakery products were tested limiting thegeneral applicability of their data to a broader range of foodproducts

Many factors play a role in detectability of allergensby immunoassays Limiting factors may be epitope recog-nition solubilityextractability grinding efficiency and in-homogeneity of the food products [28] Because it is notknown which antigens were used to prepare the antibodiesapplied in the commercial ELISAs differences in antibodyspecificity may also affect the recovery With regard to

epitope recognition we use two independent sources ofpolyclonal antibody recognizing multiple proteins in sesameseed (Figure 1) and probably multiple epitopes per proteinas well This reduces the risk of poor recognition when aspecific part of a protein is affected or damaged duringfood processing Solubility and extractability depend on thematrix and the extraction buffer Apparently our methodworks well for tahini although this is a high-fat matrixGrinding and inhomogeneity of the samples may be an issuefor bakery products where whole sesame seeds are oftenused as topping Preliminary data from our lab showed thatdifferent grinding procedures result in different extractionyields and recoveries in our ELISA this is a topic of furtherinvestigations The poor recovery from bakery products mayalso be the result of limited solubilityextractability and this


may be the reason that our assay shows a poor recoverywhen testing bakery products (Table 2) Baking is known todecrease the solubility and extractability of proteins leadingto low recoveries in ELISAs [30 31] Some manufacturersof commercial immunoassays to detect allergens in foodproducts add an extraction additive to improve extractabilityfrom difficult matrices like chocolate or baked productsOptimizing grinding and extraction proceduremay be usefulto further develop our ELISA The two commercially avail-able assays we evaluated in this study also show remarkabledifferences in recovery which were in some cases relativelylow It may turn out that certain food products shouldpreferably be analyzed with the one assay while other typesof food product require another assay for optimal analysis

4 Concluding Remarks

A set of polyclonal antibodies for the detection of sesame seedprotein was developed The sandwich ELISA constructedwith these antibodies was sensitive and specific for thedetection of sesame seed residues However the recoveryfrom baked food products was low Other assays appear tohave better recoveries for certain food products The currentdata show that different assays may be needed to measuresesame seed residues reliably in different food products andthat great care should be taken when assessing the level ofsesame seed residues in food products

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors wish to acknowledge the leadership role of thelate Professor Susan Hefle in this research project ProfessorHefle initiated this research project and led the design of theexperimental approaches

References

[1] L Zuidmeer K Goldhahn R J Rona et al ldquoThe prevalence ofplant food allergies a systematic reviewrdquo Journal of Allergy andClinical Immunology vol 121 no 5 pp 1210ndash1218 2008

[2] S H Sicherer A Munoz-Furlong J H Godbold and H ASampson ldquoUS prevalence of self-reported peanut tree nut andsesame allergy 11-year follow-uprdquo Journal of Allergy andClinicalImmunology vol 125 no 6 pp 1322ndash1326 2010

[3] D Aaronov D Tasher A Levine E Somekh F Serour and IDalal ldquoNatural history of food allergy in infants and children inIsraelrdquo Annals of Allergy Asthma and Immunology vol 101 no6 pp 637ndash640 2008

[4] I Dalal I Binson R Reifen et al ldquoFood allergy is a matterof geography after all sesame as a major cause of severe IgE-mediated food allergic reactions among infants and youngchildren in Israelrdquo Allergy vol 57 no 4 pp 362ndash365 2002

[5] D Dano B C Remington C Astier et al ldquoSesame allergythreshold dose distributionrdquo Food and Chemical Toxicology vol83 pp 48ndash53 2015

[6] M N Kolopp-Sarda D A Moneret-Vautrin B Gobert et alldquoSpecific humoral immune responses in 12 cases of foodsensitization to sesame seedrdquoClinical and Experimental Allergyvol 27 no 11 pp 1285ndash1291 1997

[7] K Beyer L Bardina G Grishina and H A Sampson ldquoIdenti-fication of sesame seed allergens by 2-dimensional proteomicsand Edman sequencing seed storage proteins as common foodallergensrdquo Journal of Allergy and Clinical Immunology vol 110no 1 pp 154ndash159 2002

[8] V Leduc D A Moneret-Vautrin J T Tzen M Morisset LGuerin and G Kanny ldquoIdentification of oleosins as majorallergens in sesame seed allergic patientsrdquo Allergy vol 61 no3 pp 349ndash356 2006

[9] C Magni C Ballabio P Restani et al ldquoMolecular insight intoIgE-mediated reactions to sesame (Sesamum indicum L) seedproteinsrdquoAnnals of Allergy Asthmaamp Immunology vol 105 no6 pp 458ndash464 2010

[10] C Alonzi P Campi F Gaeta F Pineda and A RomanoldquoDiagnosing IgE-mediated hypersensitivity to sesame by animmediate-reading lsquocontact testrsquo with sesame oilrdquo Journal ofAllergy and Clinical Immunology vol 127 no 6 pp 1627ndash16292011

[11] F T Husain I E Bretbacher A Nemes and M Cichna-Markl ldquoDevelopment and validation of an indirect competitiveenzyme linked-Lmmunosorbent assay for the determinationof potentially allergenic Sesame (Sesamum indicum) in foodrdquoJournal of Agricultural and Food Chemistry vol 58 no 3 pp1434ndash1441 2010

[12] G Redl F THusain I E Bretbacher ANemes andMCichna-Markl ldquoDevelopment and validation of a sandwich ELISA forthe determination of potentially allergenic sesame (Sesamumindicum) in foodrdquo Analytical and Bioanalytical Chemistry vol398 no 4 pp 1735ndash1745 2010

[13] K Schoringhumer G Redl and M Cichna-Markl ldquoDevel-opment and validation of a duplex real-time PCR methodto simultaneously detect potentially allergenic sesame andhazelnut in foodrdquo Journal of Agricultural and Food Chemistryvol 57 no 6 pp 2126ndash2134 2009

[14] A Ehlert A Demmel C Hupfer U Busch and K-H EngelldquoSimultaneous detection of DNA from 10 food allergens byligation-dependent probe amplificationrdquo Food Additives andContaminants Part A Chemistry Analysis Control Exposureand Risk Assessment vol 26 no 4 pp 409ndash418 2009

[15] R Schade C Staak C Hendriksen et al The Production ofAvian (Egg Yolk) Antibodies IgY The Report and Recommen-dations of ECVAM Workshop 21 (22ndash24 March 1996 BerlinGermany) European Center for the Validation of AlternativeMethods (ECVAM) Ispra Italy 1996 ECVAM TP 580 JRCEnvironmental Institute Ispra Italy

[16] S L Hefle E Jeanniton and S L Taylor ldquoDevelopmentof a sandwich enzyme-linked immunosorbent assay for thedetection of egg residues in processed foodsrdquo Journal of FoodProtection vol 64 no 11 pp 1812ndash1816 2001

[17] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[18] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[19] H Towbin T Staehelin and J Gordon ldquoElectrophoretic trans-fer of proteins frompolyacrylamide gels to nitrocellulose sheetsprocedure and some applicationsrdquo Proceedings of the National


Academy of Sciences of the United States of America vol 76 no9 pp 4350ndash4354 1979

[20] S L Taylor D A Moneret-Vautrin R W R Crevel et alldquoThreshold dose for peanut risk characterization based upondiagnostic oral challenge of a series of 286 peanut-allergicindividualsrdquo Food and Chemical Toxicology vol 48 no 3 pp814ndash819 2010

[21] B K Ballmer-Weber M Fernandez-Rivas K Beyer et al ldquoHowmuch is too much Threshold dose distributions for 5 foodallergensrdquo The Journal of Allergy and Clinical Immunology vol135 no 4 pp 964ndash971 2015

[22] S L Taylor J L Baumert A G Kruizinga et al ldquoEstablishmentof reference doses for residues of allergenic foods report of theVITAL expert panelrdquo Food amp Chemical Toxicology vol 63 pp9ndash17 2014

[23] G Kanny C De Hauteclocque and D A Moneret-VautrinldquoSesame seed and sesame seed oil contain masked allergens ofgrowing importancerdquo Allergy vol 51 no 12 pp 952ndash957 1996

[24] B Eberlein-Konig F Rueff and B Przybilla ldquoGeneralized ur-ticaria caused by sesame seeds with negative prick test resultsand without demonstrable specific IgE antibodiesrdquoThe Journalof Allergy and Clinical Immunology vol 96 no 4 pp 560ndash5611995

[25] L Caminiti D Vita G Passalacqua et al ldquoTahini a littleknown sesame-containing food as an unexpected cause ofsevere allergic reactionrdquo Journal of Investigational Allergologyand Clinical Immunology vol 16 no 5 pp 308ndash310 2006

[26] K A B M Peeters A C Knulst F J Rynja C A F MBruijnzeel-Koomen and S J Koppelman ldquoPeanut allergysensitization by peanut oil-containing local therapeutics seemsunlikelyrdquo The Journal of Allergy and Clinical Immunology vol113 no 5 pp 1000ndash1001 2004

[27] S J Koppelman R Vlooswijk G Bottger et al ldquoDevelopmentof an enzyme-linked immunosorbent assay method to detectmustard protein inmustard seed oilrdquo Journal of Food Protectionvol 70 no 1 pp 179ndash183 2007

[28] U Immer ldquoFactors affecting the effectiveness of allergen detec-tionrdquo in Detecting Allergens in Food S J Koppelman and S LHefle Eds CRC PressWoodhead Publishing Cambridge UK2006

[29] R E Poms M E Agazzi A Bau et al ldquoInter-laboratoryvalidation study of five commercial ELISA test kits for the deter-mination of peanut proteins in biscuits and dark chocolaterdquoFood Additives and Contaminants vol 22 no 2 pp 104ndash1122005

[30] S L Taylor J A Nordlee LMNiemann andDM LambrechtldquoAllergen immunoassaysmdashconsiderations for use of naturallyincurred standardsrdquo Analytical and Bioanalytical Chemistryvol 395 no 1 pp 83ndash92 2009

[31] M L Downs and S L Taylor ldquoEffects of thermal processing onthe enzyme-linked immunosorbent assay (ELISA) detection ofmilk residues in a model food matrixrdquo Journal of Agriculturaland Food Chemistry vol 58 no 18 pp 10085ndash10091 2010

Submit your manuscripts athttpwwwhindawicom

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

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


are introduced in the diet at an early age as a good source ofcalories and bioavailable iron Thus sensitization to sesameseed and clinical sesame seed allergy before age of 2 years isdocumented [4]

Allergic reactions to the ingestion of sesame seed can betriggered by doses as low as 1mg of sesame seed protein forindividual cases and the population threshold study showedthat approximately 5mg of sesame seed protein triggers areaction in 10 of the sesame seed-allergic population [5]Sesame seeds contain several allergenic proteins that havebeen identified and partially characterized [6ndash9] Sesame oilcan also present a hazard to sesame seed-allergic individualsas it is often not highly refined and thus contains proteinresidues [10]

Unintentional ingestion of sesame seed is a risk forsesame seed-allergic consumers The presence of sesameseeds and ingredients derived from sesame seeds is not alwaysobvious Sesame seed flour paste and oil are not easilyidentified if products are not appropriately labelledThe seedsare small and a few seeds can easily be indistinguishable in abakery formulation Sesame seed residues can also occur infoods from cross-contact through use of shared processingequipment and facilities A need exists for analytical methodsthat are able to sensitively and specifically detect residuesof sesame seed Immunoassays for the detection of sesameseed proteins [11 12] and PCR methods for the detection ofDNA fragments [13 14] from sesame seed have previouslybeen described and commercial immunoassay kits exist forthe detection and quantification of sesame seed residuesPreliminary data from our group indicated that the variousmatrices in which sesame seed can potentially be presentcomplicated the quantification of sesame seed residues infood products While some methods seem appropriate forbakery products others may be more suitable for tahini Theaim of this study is to develop an ELISA for the detectionand quantification of sesame seed residues in foods andto compare its performance with regard to sensitivity andrecovery from various food matrices with two commerciallyavailable immunochemical assays for sesame seed

2 Materials and Methods

21 Immunogen Preparation Black (India black) and white(Mexico white hulled) sesame seeds were purchased fromPenzeys Ltd (Penzeys Spices Madison WI) and stored atroom temperature until needed Sesame seeds were washedthoroughly 1 8 (wv) with distilled water 6 times and driedunder a fume hood After drying the seeds were groundto uniform consistency using a blender (Oster ProfessionalProducts Chicago IL) Ground white and black sesameseeds were mixed and defatted by washing with ethyl ether(1 5 wv) with continuous stirring for 5 minutes the processwas repeated 5 timesThedefatted residuewas then allowed tosettle for 30 minutes and after that time the supernatant wasdiscarded At the last washing step the mixture was collectedusing a Buchner funnel fitted with Whatman number 1 filterpaper (Whatman International Ltd Maidstone England)and a final washing of the defatted sesame seeds was donetwice with acetone (1 5 wv) The defatted material was then

dried under a fume hood at room temperature for 48 hoursThis material was used for the immunization of animals Thecrude protein content of the material was determined usingthe Kjeldahl method Immunogen was prepared by emulsify-ing the dried material with adjuvant rather than first makingan extract in order to target the largest possible diversity ofepitopes including those from lipophilic proteins CompleteFreundrsquos adjuvant (CFADifco LabsDetroitMI)was used forpreparting the primary immunogen and incomplete Freundrsquosadjuvant (IFA) (Difco Labs Detroit MI) was used to prepareimmunogen for booster immunizations

22 Polyclonal Antibody Production One goat was immu-nized with 10mg of defatted sesame seed immunogenemulsifiedwithCFAand administrated subcutaneously (sc)The first booster injection comprised of 500120583g sesame seedimmunogen in IFA was administered 2 weeks after the initialimmunization Subsequent booster injections were admin-istered with 250120583g of defatted sesame seed in IFA every 3weeks after that alternating between sc and intramuscularly(im) The first test bleed was taken after the second boosterinjection (approximately 5 weeks after the initial injection)and thereafter test bleedswere taken 10 days after each boosterinjection

Three chickens were initially immunized with defattedsesame seed immunogen in CFA followed by booster injec-tions at 2-week intervals using 200120583g in IFA (sc and imalternatively) basically according to Schade et al [15] Eightweeks after the initial immunization this group was restedfor approximately 6 weeks and then booster injections werechanged to 3-week intervals due to an observed decrease inantibody production

Titer development was monitored using a noncompet-itive ELISA basically as described by Hefle et al [16] Inshort microtiter plates (MaxiSorp Nagle Nunc InternationalRoskilde Denmark) were coated with 1 120583g sesame seedproteinmL of coating buffer (0015M Na

2CO3 0035M

NaHCO3 and 002NaN

3 pH 96) and incubated overnight

at 4∘C After blocking with 350 120583L of PBS containing 01gelatin (porcine 300 bloom Sigma Chemical Co St LouisMO) sera from goat or chicken egg yolks were incubatedin various dilutions Plates were washed with PBS con-taining 005 Tween 20 and bound goat IgG and eggIgY were detected and quantified using commercial anti-immunoglobulin-alkaline phosphatase conjugates (anti-goatIgG [Pierce Chemical Co Rockford IL] anti-chicken IgY[Promega Co Madison WI]) respectively following themanufacturerrsquos instructions Bleeds from the goat with titersabove 10000 were pooled Yolks with titers gt3000 werepooled

23 Antibody Isolation Polyclonal IgG antibodies were par-tially purified from the sera by precipitation in 50 and35 ammonium sulfate [16] The partially purified IgG wasbriefly dialyzed against deionized water and then extensivelydialyzed against 001M phosphate-buffered saline (PBS)(0002M NaH

2PO4 0008M Na

2HPO4 085 NaCl and

002 NaN3 pH 74) as described by Hefle et al [16] This

IgG fraction was used as the coating antibody for ELISA






2CO3 0035M NaHCO

3 and



















MW



WS BS

200

21

14

65

45

97

37



Abso

rban

ce at

410

nm

00

05

10

15

20

25













Table 1 Continued











Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2CO3 0035M NaHCO

3 and



















MW



WS BS

200

21

14

65

45

97

37



Abso

rban

ce at

410

nm

00

05

10

15

20

25













Table 1 Continued











Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























MW



WS BS

200

21

14

65

45

97

37



Abso

rban

ce at

410

nm

00

05

10

15

20

25













Table 1 Continued











Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MW



WS BS

200

21

14

65

45

97

37



Abso

rban

ce at

410

nm

00

05

10

15

20

25













Table 1 Continued











Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























Table 1 Continued











Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table 1 Continued











Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Middle part


Lighter crust end


Darker crust end















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgment


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Sandwich Enzyme-Linked Immunosorbent Assay for Detecting Sesame …downloads.hindawi.com/journals/bmri/2015/853836.pdf · 2019. 7. 31. · Immunogen Preparation