Journal of Immunological Methods 373 (2011) 247–251

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Journal of Immunological Methods

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Technical note

A phage display system for the identification of novelAnisakis simplex antigens

Itziar López, Miguel Angel Pardo⁎Food Research Division, AZTI-Fisheries and Food Technological Institute, Derio, Spain

a r t i c l e i n f o

⁎ Corresponding author at: Food Research DivisioFood Technological Institute, Parque Tecnológico de BEdificio 609, E-48160Derio (Bizkaia), Spain. Tel.:+34 994 657 25 55.

E-mail address: mpardo@azti.es (M.A. Pardo).

0022-1759/$ – see front matter © 2011 Elsevier B.V.doi:10.1016/j.jim.2011.08.009

a b s t r a c t

Article history:Received 21 July 2011Received in revised form 4 August 2011Accepted 11 August 2011Available online 27 August 2011

Anisakis simplex has been recognized as an important cause of disease in man and as afoodborne allergen source. Actually, this food-borne was recently identified as an emergingfood safety risk including allergenic symptoms. This parasite contains a large variety ofallergenic proteins enforcing the necessity to detect new allergens. Commonly, these effortshave been focused on the developing of cDNA libraries, where virtually all expressed mRNAsare present, by using immunoreactive patient serum or polyclonal antibodies. Phage displaysystem is an alternative strategy which permits the physical binding of the genotype with thephenotype, since the products are expressed by the phage on its surface, thereby allowingmoreefficient selection. In this work we have constructed two libraries in the pJuFo phage, obtaininga primary titer of around 103 cfu/ml and an amplified titer of the order of 1013 cfu/ml whereasthe insert sizes varied from 0.35 to 1.2 kb. Both libraries were subsequently analyzed byenrichment with polyclonal antibodies to an A. simplex extract and immunoreactive sera frompatients with a clinical history of allergy to this parasite. Finally, 30 clones were scrutinizeddetecting several Anisakis candidate antigens. Actually, one protein, belongs to the fructose-1,6-bisphosphatase family, was found in 34% of scrutinized clones revealing as a promisingnovel A. simplex allergen. Phage display technology has to date not yet been applied to theidentification of new A. simplex allergens, and the present work opens up new avenues to theunderstanding of the Anisakis allergenic process.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Anisakis simplexAllergensPhage displaypJuFo

1. Introduction

Anisakis simplex is a parasitic helminth which belongs tothe nematode class and Anisakidae family. Third stage larvae(L3) parasite crustaceans and copepods. These invertebratesare then ingested by cephalopods (e.g. squids) and fish whichmaintain the L3 larvae. The life cycle is completed whenmarine mammals (Cetaceans), or accidentally humans, eatthese infected animals and thus become their final host.

The presence of A. simplex and of other nematodes inhumans has increased substantially over the last two decades.In fact, A. simplex is the nematode which is most frequently

n, AZTI-Fisheries andizkaia, Astondo Bidea-4 657 40 00; fax:+34

All rights reserved.

associated with human infections. Generally, this infectionarises from the consumption of raw or incompletely cooked fish(Audicana and Kennedy, 2008). In such cases, the ingestion oflive larvae generates the disease known as Anisakiasis, whichinvolves nausea and intense abdominal pain. Another of thepathologies produced by A. simplex is due to its capacity toinduce allergic and/or anaphylactic reactions. In fact, thisnematode has recently been identified as an emergent riskaccording to the report publishedby the EuropeanCommunity'sRapid Alert System for Food and Feed (RASFF).

To date, A. simplex presents 12 allergens that most of themhave been recently reported (Pérez-Pérez et al., 2000;Kobayashi et al., 2007; Rodríguez et al., 2008; Caballero et al.,2011; Kobayashi et al., 2011). In fact, it has been postulated thatsomatic extracts of A. simplex contain a large variety ofallergenic proteins and that the sensitivity and reactivity ofpatients to these allergens is highly variable. Using SDS-PAGEor

248 I. López, M.A. Pardo / Journal of Immunological Methods 373 (2011) 247–251

immunoblot with reactive serum from patients, new allergenscan be identified, without knowing their amino acid or nucleicacid sequences. Another method to identify novel allergensconsists in the creation of a cDNA library in which virtually allcDNA copies of all the mRNA is present. From this, one canidentify the proteins via immunological screening or via thetechnique called phage display.

In contrast to cDNA library immunological screening, thephage display technique has the advantage of not requiringscreening via immobilization of phages to nitrocellulose or PVDFmembranes (Crameri, 1997). Rather, this technique permits thephysical bindingof thegenotypeandphenotype, bymeansof thebinding of expressed proteins on the surface of the phage. One ofthemainvectors used for the identificationof allergens via phagedisplay is the pJuFo vector, which permits the binding to thephage of the expression product via the use of leucine zippers. Infact, this phage has been successfully used for the identificationof various allergens of fungi, mites, wheat gliadins and peanut.

In thepresentwork,wehave constructed two cDNA librariesin the pJuFo phage, which have been used to identify a numberof antigens of the A. simplex parasite. Following a number ofenrichment stages and screening with immunoreactive serafrompatients with a clinical history of allergy to this parasite, aswell aswith polyclonal antibodies toA. simplex somatic extracts,we have identified a variety of antigens which are novelcandidate allergens. These will have to be tested against morepatient sera, in order to definitively confirm that they areeffectively new allergens. In any event, this pJuFo A. simplexlibrary opens up new avenues of research for the identificationof new allergens for this parasite.

2. Materials and methods

2.1. A. simplex L3 larvae

Live L3 larvae of A. simplexwere obtained bymeans of directextraction from viscera of Micromesistius poutassou (bluewhiting), as reported by López and Pardo (López and Pardo,2010).

2.2. Patient serum

Sera samples were obtained from patients who had aclinical history of A. simplex hypersensitivity and presentedwith a level of Anisakis-specific IgE antibodies higher than0.35 kU/l. Anisakis-specific IgE antibody levels were measuredusing the Pharmacia CAP System (Phadia, Uppsala, Sweden).The extraction of serum from human patients was approved bythe Ethics Board of Santiago Hospital (Vitoria-Gasteiz, Spain).

2.3. Construction of a cDNA library in the pJuFo phage

We initially constructed a cDNA library in theλ phage frommRNA from L3 A. simplex larvae, using the ZAP-cDNA GigapackIII Gold Cloning Kit (Stratagene, Agilent Technologies, Inc.Santa Clara, USA). The cloning vector UNI-ZAPXRwas used forthe development of the library.mRNAwas isolated from1 g ofA. simplex tissue by means of affinity purification in oligo-dTcellulose columns using the FastTrack™ 2.0 commercial kit(Invitrogen Dynal AS, Oslo, Norway). The concentration andpurity of the resulting mRNAwere determined by spectropho-

tometry using a GeneQuant™ PRO Spectrophotometer (GEHealthcare Bio-Sciences AB, Uppsala, Sweden), by measuringabsorbance at 260 nm and the 260/280 absorbance ratios,respectively. cDNA was synthesized from 5 μg of mRNA. Theobtained cDNA was subjected to cDNA fractioning usingCHROMA SPIN 200 TE columns (Clontech Laboratories Inc.,Mountain View, USA). Ligation of the UNI-ZAP XR cloningvector was carried out for 48 h at 4 °C, following thespecifications of the manufacturer. Packaging was carried outby adding 4 μl of the ligation mixture and incubating for 2 h at22 °C. The primary librarywas titrated and amplified accordingto themanufacturer's specifications. Oncewe had obtained thiscDNA library in theλphage,weperformed its in vivoexcision toobtain the pBluescript phagemid. The phagemid DNA wasprepared using the UltraCleanTMMaxi Plasmid Prep Kit (MoBioLaboratories, Carlsbad CA) following the specifications of themanufacturer. The inserts (larger than 200 bp) were purifiedafter digestion using the XbaI and KpnI enzymes.

In order to construct the library in the pJuFo vector, weligated the inserts which had been cut from the λ phagelibrary with the pJuFo vector which had previously beendigested with the XbaI and KpnI enzymes. Ligation conditionswere 1:1.5 vector:insert in the presence of the T4 DNA ligaseenzyme at 14 °C overnight. Once ligation had been carriedout, ECM399BTX equipment (Harvard Apparatus, Hollliston,USA) was employed for the electroporation of XL1 electro-competent cells (Stratagene, Agilent Technologies, Inc. SantaClara, USA) at 2500 V. Next 3 ml SB medium was added andthe electroporated solutionwas incubated for 1 h at 37 °C.Wesubsequently adding SB medium, supplemented with 1 μg/mlampicillin to a final volume of 10 ml. Transformed cells weretitrated by plating in LB medium supplemented with 1 μg/mlampicillin and 0.15 μg/ml tetracycline. The remaining solu-tion was incubated for 60 min at 37 °C. Then ampicillin wasadded to a final concentration of 50 μg/ml and the solutionincubated again for 1 h at 37 °C. Following incubation, weadded 1012 pfu of the VCSM13 helper phage (Stratagene,Agilent Technologies, Inc. Santa Clara, USA). The temperaturewas maintained at 37 °C for 5 min and then for 2 h at 37 °C inSB medium supplemented with 1 μg/ml ampicillin and0.15 μg/ml tetracycline. Two hours later, we added kanamycinto a final concentration of 70 μg/ml and incubated the mixovernight at 31 °C. In order to precipitate the phage, the culturewas centrifuged at 5000 g for 15 min at 4 °C; the phage-containing supernatantwas recovered and to this we added 4%(w/v) polyethylene glycol 8000 and 3% (w/v) NaCl. Thissolution was maintained at 4 °C for 2 h to permit theprecipitation of the phage and then centrifuged at 8000 g for20 min at 4 °C. Finally, the supernatant was discarded and thepellet was resuspended in PBS buffer with 1% (w/w) bovineserum albumin (BSA). The phage was titrated by means of theaddition of 1 μl of dilutions between10−3 and 10−8 to 200 μl ofXL1 cells (OD600: 0.5). Once titrated, the phage was conservedat−20 °C. We obtained two cDNA libraries in the pJuFo vector(B1 and B2). The insert size was measured by analyzing in anagarose gel the fragments amplified by PCR.

2.4. Enrichment and analysis of clones by immunoaffinity

Affinity enrichment of the B1 library was performed usingpolyclonal antibodies (EA1) to a commercial extract of A.

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simplex (ALK Abelló, Hørsholm, Denmark). The EA1 antibodieswere obtained from rabbits injected subcutaneously ondifferent sites with the A. simplex extract. Evaluation of positivereaction of the sera, purification and titration of polyclonalantibodies was carried out in Abintek Biopharma S. L (Derio,Spain). Analysis of clones from the B2 library was performedusing amixture of sera frompatients. In both cases, enrichmentwas carried out following the protocol reported by Crameri(1997) with modifications: 3 wells of an ELISA plate werecoveredwith 150 μl of a 10% dilution of EA1 antibody in elutionbuffer (100 mM glycine HCl, pH 2.2) and incubation for 2 h at37 °C. Non-adhering antibodywas eliminatedby2washeswithPBS. In the case of patient serum, 3wells of an ELISA plate werecovered with 100 μl anti-human IgE (0.5 mg/ml; ZymedInvitrogen Dynal AS, Oslo, Norway) for 2 h at 37 °C. Subse-quently,wellswerewashed twicewith PBS,filledwith blockingsolution and incubated for 1 h at 37 °C. Then, the blockingsolution was removed and we added to each well 30 μl of theserummixwith 70 μl of recovering buffer and incubated for 2 hat 37 °C. Wells were then washed twice with PBS to eliminateunbound antibody. Once the relevant antibodies had bound tothe plaques, the same protocol was employed for both cases.Wells were filled with blocking solution, incubated for 1 h at37 °C, washed twicewith PBS and the phagewas added to eachwell which was incubated for 2 h at 37 °C. After incubation,non-bound phage was removed by washing with water, 10washes in PBS supplemented with 5% (v/v) Tween 20, 10washeswith PBS and twowasheswithwater. Boundphagewaseluted by adding 150 μl elution buffer and incubating for10 min at room temperature. The eluate was recovered bypipetting, transferring it to 18 μl Tris 2 M (pH=10.5) for each150 μl eluate. Eluted phage was used to infect 4 ml of freshlyobtained XL1 cells (OD600=0.5), by means of incubation for15 min at 37 °C. SBmediumwas added,which contained1 μg/mlampicillin and0.15 μg/ml tetracycline andprewarmedat 37 °C toafinal volumeof10 ml. Subsequently,weplated20,10and1 μl inLB-containing dishes in the presence of 1 μg/ml ampicillin and0.15 μg/ml tetracycline, incubating at 37 °C overnight to obtainthe titer of eluted phagemids. The remaining culture wasincubated for 1 h at 37 °C; ampicillin was added at a finalconcentration of 50 μg/ml and incubation proceeded again foranother hour. We then added 1012 pfu VCSM13 helper phage tothe culture whichwasmaintained at 37 °C for 5 min, topping upthe volume to 100 ml with SB supplemented with 1 μg/mlampicillin and 0.15 μg/ml tetracycline, incubating for 2 h at 37°.After 2 h, we added kanamycin at a final concentration of70 μg/ml and incubation continued overnight at 31 °C. Toprecipitate the phage, we centrifuged the mix at 5000 g for15 min at 4 °C. The phage-containing supernatant wasrecovered and 4% (v/v) PEG8000 and 3% (v/v) NaCl wereadded. This solution was maintained at 4 °C for 2 h toprecipitate the phage. Then we centrifuged at 8000 g for20 min at 4 °C and the supernatantwas discarded. The resultingpellet was resuspended in PBS with 1% (w/v) BSA. The phagewas then titrated by means of the addition of 1 μl of dilutionsbetween 10−3 and 10−8 to 200 μl of XL1 cells (OD600=0.5).Once the phage was titrated, it was conserved at−20 °C.

The percentage of recovery rate of successive rounds ofenrichment was determined as the coefficient of the numberof eluted phages to the number of applied phages. In contrast,fold-enrichment was measured as the coefficient of the

recovery rate of each round of enrichment and the recoveryrate produced in the first enrichment round. Finally, enrichedclones were analyzed by PCR and PCR amplicons weresequenced according to the conditions reported by Kemp etal. (2002). Obtained sequences were analyzed using Blastsoftware (http://www.ncbi.nlm.nih.gov).

3. Results and discussion

Progress over the last decade with the techniques ofmolecular biology has contributed to the development of thefield of molecular allergology. The necessity to detect newallergens has stimulated the development of methods for theimmunoscreening of cDNA libraries. In order to identify newallergens, it is necessary to create a cDNA library in whichvirtually all cDNA copies of all expressed mRNAs are present.Once the library has been constructed, immunoscreening can beperformed using both immunoreactive patient serum andpolyclonal antibodies obtained fromextracts. Immunoscreeningis typically performed after transferring phages to nitrocellulosemembranes. In fact, all reported cases in which new A. simplexallergens have been discovered were performed in this way. Inthe case of paramyosin (Ani s 2), screening employed the serumof a rabbit immunizedwith an A. simplex extract (Pérez-Pérez etal., 2000). The Ani s 5, Ani s 6 and Ani s 7 allergens were alsoidentified by means of this technology using a patient serum(Kobayashi et al., 2007) andmonoclonal antibody (Rodríguez etal., 2008). Finally, the allergenic proteins Ani s 10, 11 and 12were detected after membrane incubation with serum from apatient (Caballero et al., 2011; Kobayashi et al., 2011). However,one of the principal limitations of this screening technique is theneed to immobilize the phages to the membrane (Crameri,1997). Thismakes screeningdifficult andhinders the recoveryofthe relevant phage. Moreover, expressed proteins can loseantigenicity during immobilization to the membrane (Crameri,1997). In addition, immunodetection on a membrane has lowsensitivity when conventional detection methods are used;nevertheless, these can be improved with more powerfuldetection systems, such as chemiluminescence (Caballero etal., 2011; Kobayashi et al., 2011). An alternative to membraneimmunodetection is the technique knownas phage display. Thispermits the physical binding of the genotype with thephenotype, since the products are expressed by the phage onits surface, thereby allowing more efficient selection. Thepresence of stop codons in the 3´ end of cDNA makes bindingto the phage impossible, since the phage pIII protein binds to itsamino terminal end. To overcome this problem, we used thepJuFo vectorwhich allows the bindingof the expression productto the phage via leucine zippers. In fact, this phage has alreadybeen successfully used to identify various allergens fromdifferent sources.

Here, we have constructed two libraries (B1 and B2) in thepJuFo phage from the library obtained in theλ phage, obtaininga primary titer of 103 cfu/ml and an amplified titer of the orderof 1013 cfu/ml. Insert sizes varied from 0.35 to 1.2 kb. Once thelibrary had been amplified, the B1 library was enriched usingEA1 polyclonal antibodies to a commercial extract of A. simplex,whereas the B2 librarywas enriched using a patient serummix.We carried out 4 enrichments of the B1 library and observed anincrease in recovery rate in each of the rounds, as well as a risein the increase produced in each amplification round (Table 1).

Table 1Affinity enrichment of the two cDNA libraries in the pJuFo phage. The B1 library was enrichedwith EA1 antibodies, whereas the B2 library was enrichedwith a poolof patient serum.

Library Enrichment round Phage applied (pfu) Phage eluted (pfu) Amplified Recovery rate Enrichment (fold)

B1 1 2×1012 3.0×103 1.4×1012 1.5×10−7 1.02 1.4×1011 7.4×102 6.9×1012 5.2×10−7 3.463 6.9×1011 7×103 1.4×1014 1.01×10−6 6.734 1×1011 5×105 3.2×1011 5×10−4 3333

B2 1 8,9×1011 2,3×103 7,1×1012 2,58×10−7 1.02 7,1×1011 8×105 1×1012 1,12×10−4 434.13 1×1011 3.1×105 9×1011 3,1×10−6 12.0

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In the case of the B2 library, we performed 3 enrichments, witha remarkably increase in the first round, while in the secondround, there was no increase. A substantial increase inenrichment was found during the first two rounds, whilerecovery rate did not increase during the third enrichmentround. Thesedata are indicativeof thehigh recovery rate of eachenrichment round (Table 1).

Then we analyzed 15 clones from each of the final enrichedlibraries and found a number of proteinswhichwere present inboth libraries. Once the inserts had been sequenced, we nextanalyzed themusingBlast software, obtaining the resultswhichare shown in Table 2. The results indicate that enrichment ofcertain clones took place. Of particular interest is the homologyof ten of the analyzed clones (i.e. 34%) with the sequence of theCaenorhabditis elegans protein belonging to the fructose-1,6-bisphosphatase family (NP 491004). This protein is indeed astrong candidate A. simplex allergen because one of the positiveclones was found to be IgE reactive after the enrichment withimmunized patient. In fact, this proteinwas already reported tobe a wheat flour allergen (Baur and Posch, 1998). Futureresearch should involve the subcloning and expression of thisprotein to test its allergenicity in in vitro and in vivo studies.

Similarly, we found 3 clones (10%) which share homologywith subunit III of mitochondrial cytochrome c oxidase(AY994157.1) and another 2 clones with homology with

Table 2Analysis of the homology of 30 clones randomly selected from the enrichment of t

Total number ofclones

Homologue Orga

10 Fructose-1,6-BiPhosphatase family member(fbp-1)

Caenelega

3 Cytochrome c oxidase subunit III Anisa2 mRNA for nicotinic acetylcholine receptor alpha

subunitAscar

2 Cellulose binding protein precursor cbp-1 Meloincog

2 24 kDa secreted protein mRNA Brugi1 Hypothetical protein (10 or fewer PubMed links) Brugi1 Mitochondrion, complete genome NADH

dehydrogenase subunit 4 LAnisa

1 hypothetical protein F40A3.3 Blast x Caenelega

1 Mitochondrion, complete genome Cytochrome b Anisa1 Hypothetical protein CBG18160 Caen

brigg6 No h

theα subunit of the nicotinic acetylcholine receptor of Ascarissuum (AJ011382.1) (Table 2). Both antigens also reacted withIgE, but, to date, these proteins have not been reported to haveallergenic properties. Moreover, in the library enriched withpatient serum, we found a 24 kDa secreted protein in twoIgE-reactive clones and four antigens. All of thesemolecules arenovel candidate allergens and should be examined in in vitroand in vivo assays for potential allergenicity. A. simplex is nowbelieved to express awide array of antigenswhich can induce ahighly variable response in humans, in terms of sensitivity andreactivity. Finally, it should be noted that 20% of tested clones(n=6) exhibited no homology with any of the sequences inGenBank. Thus, the proteins selected in this work representinteresting candidate A. simplex antigens and future applica-tions of this and other technologies may reveal even moreallergenic proteins of clinical interest.

In conclusion, we have developed a cDNA library in the λphage which contains virtually all cDNA copies of all themRNA present in the A. simplex nematode. This library can beused for the identification of A. simplex to highlight thepathogenic mechanisms of the nematode, and to developingtherapeutic agents for the treatment of A. simplex-relatedpathologies. We have also constructed this cDNA library inthe pJuFo phage which has facilitated the identification of tenproteins which are recognized by A. simplex reactive patient

he B1 (IgG-mediated) and B2 (IgE-mediated) libraries.

nism GenBank Number ofIgG-positive clones

Number ofIgE-positive clones

orhabditisns

NM_058603.3 9 1

kis simplex AY994157.1 2 1is suum AJ011382.1 1 1

idogynenita

AF049139.1 2 –

a malayi AF072679.1 – 2a malayi XP_001901403 1 –

kis simplex AY994157.1 – 1

orhabditisns

NP_001023904.1 – 1

kis simplex AY994157.1 – 1orhabditissae

XP_001665781.1 – 1

omology – 6

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serum, as well as with polyclonal antibodies to an A. simplexextract. The most frequently found protein was one whichbelongs to the fructose-1,6-bisphosphatase family. All of theseantigens represent new candidate A. simplex allergens, but adeeper analysis is warranted. The generation of a cDNA libraryin the λ phage, which is highly representative of the A. simplexcDNA, will allow more optimized construction of new cDNAlibraries in the pJuFo phage. Since the technique of phagedisplay has to date not yet been applied to the identification ofnew A. simplex allergens, the present work, which reportspromising preliminary results, opens up new avenues ofpotentially fruitful investigation.

Acknowledgments

We sincerely thank Drs. Víctor de Lorenzo and Luis ÁngelFernández, Microbiology Biotechnology Department, SpanishNational Biotechnology-CSIC, for providing the pJuFo vector,and Dra. Audicana, Allergy Department, Santiago Hospital,Gasteiz-Vitoria, for providing human sera. This study wassupported by the Education, University and Research Depart-ment of the Basque Government (Project RC-2009-1-58) andalso in part by a PhD Grant for the Agriculture and FisheriesDepartment of the Basque Government.

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