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Extraction of a Monospecific Coombs-Reagentfrom Chicken EggsEsteban Gutierrez Calzado', Eduardo Cruz Marie', Tomäs Samon Chavez', Ernesto LunaYazquez', Zelma Corona Ochoa' and Rüdiger Schade'ILABEX, Laboratorio de Antieuerpos y Biomodelos Experimentales, CU-Santiago de Cuba2Institute of Pharmacology and Toxicology, Medical Faculty (Chante) of the Humboldt-University, D-Berlin

SummaryDuring the last ten years the extraction of specijic antibodies(ab) from the yolk of eggs of immunised chickens is more andmore accepted as an useful alternative to the immunisation ofmammals. The subject ofthis work is the immunisation of chick-ens with human IgG and the extraction of specific anti humanIgG ab from egg yolk in order to obtain monospecijic Coombsreagent. I2 Leghorn hens (25 weeks old) were immunised withintact human IgG (INTACGLOBIN®). The chickens were immu-nised with IODflg IgG/animal once per weekfor aperiod of sev-en weeks. The highest titre was observed after the 5th immuni-sation, the following immunisations achieved no further titreincrease. The IgY purijication was performed according to themethod of Akita and Nakai (1993). The resulting IgY prepara-tion was testedfor the presence of hetero-agglutinine by meansof direct agglutination using human erythrocytes of all bloodgroups. Thereafter 58 blood donors were tested by means of di-rect or indirect Coombs-test using a reference reagent (DAKO)and a Coombs reagent isolatedfrom chicken eggs (IgY antibod-ies). No differences have been found between the results ob-tained using both Coombs reagents. Presented results show thatthere is a possibility to produce Coombs-reagent in chickens.Advantages of this method are: I) non invasive antibody sam-pling by egg collection instead of bleeding the anima I (reftne-ment of antibody production); 2) decreasing amount of animalsnecessary to produce high amounts of reagent; 3) IgY-prepara-tion contains no hetero-agglutinine in contrast to serum abfrommammals, therefore additional step in reagent production e.g.the absorption of hetero-agglutinins is not necessary.

Keywords: egg yolk ab, IgY, Coombs-reagent, IgY-technology

1 Introduction

Zusammenfassung: Gewinnung eines monospezifischenCoombs-Reagens aus dem HühnereiDie Extraktion spezijischer Antikörper (Ak) aus dem Dotter vonEiern immunisierter Hühner als tierschonende Alternative zurImmunisierung von Säugern findet in den letzten Jahrenzunehmende Akzeptanz. Ziel der vorliegenden Arbeit war dieGewinnung von monospezifischem Coombs-Reagens durchImmunisierung von Hühnern mit humanem IgG und an-schließender Extraktion der IgY-Fraktion aus dem Dotter.I2 Leghorn Hennen (25 Wochen alt) wurden mit humanem IgG(IOD tcg Intacglobinrllmmunisierung) über eine Periode vonsieben Wochen immunisiert (Ix/Woche). IgY wurde aus demDotter entsprechend der Methode von Akita und Nakai (1993)extrahiert. Der höchste Ak-Titer ließ sich nach fünf Wochenbeobachten. Testung der resultierenden IgY-Präparationen aufHeteroagglutinine mittels direkter Agglutination (alle Blut-gruppen) erbrachte negative Resultate. Anschließend wurdenBlutproben von 58 Spendern mittels direktem und indirektemCoombs-Test untersucht und mit einern Referenzserum (DAKO)verglichen. Es ergab sich eine /OO-prozentige Übereinstim-mung der Ergebnisse.Dieses Resultat eröffnet so die Möglichkeit, Coombs-Reagensin Hühnern zu produzieren. Dies hätte mehrere Vorteile:I. Coombs-Reagens ließe sich über eine tierschonende Tech-nologie gewinnen. 2. Es werden weniger Tiere zur Gewinnunggrößerer Mengen des Reagens benötigt. 3. Das vorn Huhnstammende Coombs-Reagens enthält keine Heteroagglutinine,so dass eine aufwändige Absorption des Reagens entfallenkann.

In 1945 Coombs et al. (cited after Stiteset al., 1985) immunised rabbits withhuman whole blood and obtained areagent able to bind to human serum-globulin. Since this time the termCoombs-serum is used for antibodies ofanima! origin reactive with humanglobulin. Later on, Coombs-serum was

produced by immunising mammals usingwhole blood from apparently healthyblood donors or using globulin fractionsobtained from whole blood. This proce-dure is practised up to now (Witmann,1981; Stites et al., 1985). In additionthere are reports on the use of colostrumas Coombs-reagent by immunising cowswith human whole blood. Since a fewyears monoclonal ab with respective

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specificities are obtainable (e.g. DAKO,Denmark). However, up to now there areno data with respect to a standardisationor validation of the production ofCoombs-serum. That concems aspects ofimmunisation protocols, the species ofmammals chosen, use ofadjuvants etc.(Fey et al., 1973; Witmann, 1981; Stiteset al., 1985; Anonymous, 1992). In thelast ten years the interest of many

21

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CALZADO ET AL.

scientists and enterprises engaged inantibody production is attracted by theIgY-technology as a reasonable alterna-tive to the ab-production in mammals(Schade et al., 1991; Akita and Nakai,1993; Fichtali et al., 1993; Larsson et al.,1993). The IgY-ab have some advantagesagainst the IgG-ab. Chickens produce anenormous amount of ab in contrast torabbits (during a month at least 1500 mgtotal IgY can be sampled from one layinghen compared to appr. 200 mg total IgGfrom a rabbit, Schade et al., 1997). Anti-body sampling from hens is non-invasivevia egg collection, followed by antibodyextraction from yolk. Additionally, theeggs are layed continously over a longperiod. Due to the phylogenetic distancebetween birds and mammals the immunesystem of chickens responds weIl tohighly conserved antigens which do notelicit strong immune response in mam-mals (Larsson et al., 1993; Charter et al.,1995; Warr, 1995).In the last years IgY-ab are used in

many fields of research. For instancein diagnostic of a lot of bacterial orviral diseases in human and veterinarymedicine, in detection of non-alloweddrugs in food, in qualitative and quantita-tive measurement of a lot of bioactiveproteins and peptides and, increasingly,as therapeutic/prophylactic agent in vet-erinary and human medicine to reducethe use of antibiotics (Janson et al., 1995;Reyes and Lopez, 1996; Dieguez, 1997).Since the Fe-part of IgY bears no bindingsites for mammalian complement ornatural killer cells IgY does not induce acomplement mediated cell lysis or anab-dependent cell-mediated cytotoxicity(ADCC). Due to these properties IgY-ab becomes also interesting for xeno-transplantation experiments. It could beproved, that IgY-ab with a-gal specifici-ty could protect porcine endothelial cellsfrom human complement-mediated lysisand could inhibit ADCC in vitro as well(Fryer et al., 1999).

2 Animals, materials and methods

Twelve chickens (Leghorn, 25 weeksold) were obtained from a commercialbreeder (Cuban Centre for the Produc-tion of Laboratory Animals, CENPA-

22

LAB, Havana, Cuba). Each three chick-ens were kept in cages (128 cm x 65 cm x80 cm) with water and food (special dietCM 005 Al y Co, CENPALAB, Havana,Cuba) ad libitum according to generalethical regulations in Cuba and to a greatextent to the recommendation of the 21stECVAM workshop on IgY-technology(Schade et al., 1997). The animals wereimmunised each with 100 ug intact hu-man IgG (INTACGLOBIN®, IMEFA,Cuba) once per week for seven weeks(volume 0,5-1,0 ml). The IgG-solution(phosphate-buffered saline, PBS) weremixed with Freund's complete adjuvant(FCA, first immunisation, SIGMA,Germany) or Freund's incompleteadjuvant (FIA, booster-immunisations,SIGMA, Germany) in a ratio of 3:2(IgG:FCA/FIA).Blood samples (500 ul) were obtained

weekly by puncture of the wing vein(blood sampling were performed in orderto compare the ab titre developmentin the blood and in the egg yolk, respec-tively). The eggs were collected daily,identified and stored at 4°C for furtherprocessing.The IgY of the yolk of app. 70 eggs

(eggs collected from 12 hens, 5th weekafter immunisation) was extractedaccording to the method of Akita andNakai as described previously (Akitaand Nakai, 1993). Egg yolk wasmixed with distilled water (pH 5.0 byadding HCl) in a ratio of 1:9. Thesolution was centrifuged (2.500 rpm,4°C, 30 min., Medifriger, Selecta,Spain) and the supernatant wasconcentrated by ultrafiltration (Amicon-System, cut off of the membran 100 kD,Grace, USA) to the original volume.NaCI was added to the supernatant (0.15M) and the pH was adjusted to 7.2.To avoid bacterial contamination sodiumazide was added in a concentration of0.1%. The IgY-preparations (later onindicated as IgY-reagent) were storedat 4°C.The monitoring of the anti IgG ab-titre

was performed weekly in yolk extractand serum by a c1assical immunodiffu-sion technique (agar ge1 double immu-nodiffusion test, AGIDT or Ouchterlony-technique, see Witmann, 1981 and Stiteset al., 1985). The ab-titre was calculatedas mean of twelve replicates.

Prior to the use of the anti human IgGpreparation for studying blood samplesofpatients the IgY solution was tested onthe existence of hetero-agglutinins bymeans of direct agglutination using ery-throcytes of aIl blood groups. 100 ul ofIgY preparation was added to 100 ul ofan erythrocyte preparation (2% in 0,15mol/l NaCl-solution) of each humanblood group, mixed gently and kept forone hour at room temperature. Thereafterthe mixture was centrifuged for 1 min(1000 r/min) and subsequently the pres-ence of an agglutination was assessed.Furthermore, the reactivity of the IgY-reagent (IgY-pool as described above)obtained was tested using erythrocytescoated with commercial human anti-DIgG ab (DAKO, Denmark).Blood samples of 58 patients were used

to compare the reactivity of a referenceCoombs-reagent (DAKO, Denmark) withthe reactivity of the IgY based Coombs-reagent. For this study both a direct andindirect agglutination test were used(Witmann, 1981; Stites et al., 1985).

3 Results and discussion

Monitoring the ab-titre development inchicken serum and yolk-supernatant itwas found an approximately paralleldevelopment (Fig. 1).According to other authors specific

IgY-ab are detected with a delay of 5-7days (e.g. Yamamoto et al., 1975; Wool-ley and Landon, 1995). However, thesensitivity of Ouchterlony-test used in thisstudy might be too low to demonstratesuch a delay. On the other hand, since apool of serum and yolk-supernatant, re-spectively, was used for this study, prob-ably the individual delayed occurrence ofyolk IgY could no longer be observed.Despite of the difference in the occur-rence of specific IgY-ab in chicken serumand yolk the chronological dynamic ofab-titre development was found to besimilar in both fluids.As other authors (Shimizu et al., 1988;

Akita and Nakai, 1993), which used asimilar immunisation schedule (weeklyimmunisation, 100 ug antigen/immunisa-tion/animal) we have found the highestab-titre five weeks after the first immuni-sation.

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Titre development of anti human IgG ab10

9

8

7

6

5

4

3

2

n0

0 21

days

Fig. 1: Antibody titre development of hens after immunisation with human IgG.Shown is the mean value of twelve replicates. The arrows indicate date ofimmunisation.

14 287 35 42

According to international regulations(Anonymous 1992, Docket NO.84S -0182) the titre for Coombs reagentshould be in the range of 1:16-1:64 witha free agglutination up to a dilution of1:64 (Table 1). FoIlowing the regulationmentioned above the reagent produced inthis study can be classed with group 11ofmonospecific Coombs-reagents.

It could be proved that the IgY-ab arehighly specific since the ab show no reac-tivity with erythrocytes of the blood groupsA, B, and O. That means, that the IgY-abhasthe important advantage over mammalianantibodies used so far to produce Coombsreagent and preabsorption to removehetero-agglutinine with reactivity againstblood group antigens is not necessary.

CALZADO ET AL.

Therefore, IgY Coombs-reagent ful-fills international regulations (Anony-mous, 1992) demanding no crossreactiv-ities of immunoreagents used in humandiagnostic (Table 2).In a further investigation the blood of

58 blood donors was tested for the exis-tence of erythrocyte bound IgG-ab. In or-der to investigate the reliability of theIgY-reagent a commercial Coombs-reagent (DAKO, Denmark) was used as areference. Table 3 and 4 show, that thereis no difference in the results obtainedwith both test systems, direct and indirectagglutination as well.Independent on possible improve-

ments of OUfbiomodel (concerning inparticular changes in immunisationprotocol to increase the ab titre) thenon-invasive IgY antibody samplingand the enormous amount of specificab obtainable justify the introductionof this method in order to produceCoombs-reagent. In addition, if theIgY-reagent is used instead of a mam-malian Coombs-reagent it is not neces-sary to add bovine albumin. Normally,bovine albumin is necessary as an ad-ditive to improve the immunologicalreaction and to avoid false results(Kerwick and Goldsmith, 1969; Son-nenwirth and Leonard, 1985). Conse-quently, the IgY-based Coombs-reagent is very well suited to be usedas a potential immunodiagnosticum inCuba and other countries.

Tab. 1: Agglutination of IgG-anti-D coated human erythrocytes using the IgY-reagent (IgY-pool)

Dilution of IgGanti-D ab Dilution of IgY-reagent

1/1 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 1/2048

1/1 4 4 4 4 3 3 2 2 1 +/-

1/4 3 3 3 2 2 +/-

1/16 3 3 2 2 2 +/-

1/64 2 2 +/- +/-

4 Total agglutination of the erythrocytes (one large agglutinate with bright background)3 2 or 3 large agglutinates with bright background .2 Small agglutinates of similar size and with red background

Very small but distinct agglutinates with red background+/- Very small indistinct agglutinates (unclear result)

No agglutinationThe experiment was performed in triplicate.

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Tab. 2: Reactivity of the IgY-reagent in comparison with areference Coombs-reagent (DAKO)Indirect Coombs-test

Blood-donor IgY anti-human IgG Reference-anti-human IgG

1 + +2 + +3 + +4 + +5 + +6 + +7 + +8 + +9 - -

10 - -

11 + +12 - -

13 + +14 + +15 - -

16 + +17 - -

18 + +19 + +20 + +21 - -

22 + +23 + +24 + +25 + +26 - -

27 - -

28 - -

29 - -

+ or-positive or negative agglutination reactionThe experiment was performed in triplicate.

4 Conclusions

Tab. 3: Reactivity of the IgY-reagent in comparison with areference Coombs-reagent (DAKO)Direct Coombs-test

Blood-donor IgY anti-human IgG Reference-anti-human IgG

1 + +2 + +3 + +4 + +5 + +6 - -7 - -

8 - -

9 - -

10 - -

11 + +12 - -

13 + +14 - -

15 - -

16 + +17 - -

18 + +19 + +20 + +21 - -

22 - -

23 - -

24 + +25 + +26 - -

27 - -

28 - -

29 - -+ or-positive or negative agglutination reactionThe experiment was performed in triplicate.

The immunisation protocol used in thisstudy resulted in specific ab suited to beused as a monospecific Coombs-reagent.Agglutination potency of the IgY-reagentand a commercial Coombs-reagentproved to be equal. Due to the phyloge-netic distance between birds and mam-mals the IgY-reagent has the advantagenot to contain natural hetero-agglutinins.Thus, apreabsorption by using humanerythrocytes is not necessary. In ouropinion this is an important advantage ofIgY-based Coombs-reagent. In future itshould be investigated if the same animal

model is usable also for the productionof further immunodiagnostics. There isgreat demand for such reagents in bloodbanks and imrnunohematologic laborato-ries. In addition, extensive studies arenecessary to demonstrate the usefulnessof IgY-based immunohematologicreagents in comparison with tradition alproduced reagents in order to obtain abroad acceptance. That' could be animportant step to refine the production ofa lot of immunohematological reagentsand to reduce the number of animalsnecessary to obtain the amount of abneeded in routine diagnostics andresearch.

ReferencesAkita, E. M. and Nakai, S. (1993). Com-parison of four purification methodsfor the production of immunoglobulinsfrom eggs laid by hens immunizedwith enterotoxigenic E. coli strain.1. Immun. Meth. 160, 207-214.

Anonymous, March (1992). Docket No.84S - 0182. Recommended Methods foranti human globulin evaluation.

Charter, E. A., Fichtali, J. and Lo, V. K.(1995). High Performance LiquidChromatography Analysis of egg yolkimmunoglobulin. Int. 1.B. C.1, 199-208.

Dieguez, R. (1997). Produccion deanticuerpos policlonales en gallinas.

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Purificacion de IgY. Avanees enBioteenologia moderna.BioteenologiaHabana 4, 47-50.

Fey, H., Butler, R. and Marty, F. (1973).Immunisation of pregnant cows withhuman IgG. Box. Sang. 25, 245-253.

Fichtali, J., Charter, E. A, Lo, K. V. andNakai, S. (1993). Purification of anti-bodies from industrially separated eggyolk. Journal 0/ Food Seienee 58,1282-1285.

Fryer, J. P., Firca, J., Leventhal, J. R. etal. (1999). IgY antiporcine endothelialcell antibodies effectively block hu-man antiporcine xenoantibody bind-ing. Xenotransplantation 56,98-109.

Gutierrez, C. E. J. (2000). Antieuerpos deyema de huevo (IgY) corno herramien-tas para el diagnostico y la terapeutica.Revista CENIC Cieneias Biologieas31,75-82.

Janson, A. K., Smith, C. I. and Harn-marstrom, L. (1995). Biologicalproperties of yolk immunoglobulins.Adv. Exp. Med. Bioi., 371A, 685-692.

Kerwick, J. and Goldsmith, A. (1969).Infiuence of polymer on the efficacy ofserum albumin as a potenciator of in-complete Rh aglutinin. Nature 224,510-512.

Larsson, A, Balöw, R. M., Lindahl, T. L.and Forsberg, P. O. (1993). ChickenAntibodies: Taking advantage of Evo-lution. A Review. Poultry Sei. 72,1807-1812.

Reyes, V.H. and Lopez, M. F. (1996). Unmetodo simple de obtener anticuerpospolic1onales: Elaboracion de antieuer-pos de gallina. Int. J. B. C. 15, 270-274.

Schade, R., Pfister, C., Halatsch, R. andHenklein, P. (1991). Polyc1onal IgYantibodies from chicken egg yolk. Analternative to the production of mam-malian IgG type antibodies in rabbit.ATLA 19, 403-419.

Schade, R., Staak, C., Hendriksen, C. etal. (1997). The production of avian(egg yolk) antibodies: IgY. The reportand recommendations of ECVAMworkshop 21. ATLA 24, 925-934.

Shimizu, M., Fitzsimmons, R. C. andNakai, S. (1988). Anti E. coliimmunoglobulin Y isolated from eggyolk immunized chickens as apotential food ingredient. 1. Food Sei-enee 53, 1360-1366.

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Sonnenwirth, A C. and Leonard, J.(1985). Metodos y Diagnostieos deiLaboratorio Clinieo (1009-1056).Cap. IV. Tomo 2. Segunda Edicion,Editorial Cientifico Tecnica, LaHabana, Cuba, .

Stites, D. P., Stobo, J. D., Fudenberg, H.H. and Wells J. V. (1985). InmunologiaBasiea y Clinica (349-351). Cap. IX.Quinta Edicion. Editorial CientificoTecnica. La Habana, Cuba.

Warr, w., Mayor, K. E. and Higgins D.A. (1995). IgY: c1ues to the origins ofmodern antibodies. ImmunologyToday 16, 392-398.

Witmann, F. K. (1981). Interpretaeioneliniea de las pruebas de laboratorio(213-226). Cap. VIII. SegundaEdicion. Editorial Cientifico Tecnica,La Habana, Cuba.

Woolley, J. A and Landon, J. (1995).Comparison of antibody production tohuman interleukin-ö (lL-6) by sheepand chickens. 1. Immunol. Meth. 178,253-265.

Yamamoto, H., Watanabe, H., Sato, G.and Mikami, T. (1975). Identificationof immunoglobulins in chicken eggsand their antibody activity. Jap. 1. vet.Res. 23,131-140.

Correspondence toPD Dr. Rüdiger SchadeInstitut für Pharmakologie undToxikologieDorotheenstr. 94D-I 0117 BerlinTel.: +49-30-450 525 020Fax: +49-30-450525919E-Mail: ruediger.schade@charite.de

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