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Egg Yolk Antibodies (IgY) in Routine Diagnostic Work

Christian StaakFederal Institute for Consumers Health Protection and Veterinary Medicine (BgVV), D-Berlin

SummaryAccording to Linscott's Directory of Immunological andBiological Reagents (1994/95) the commercial availabilityof egg-yolk antibodies (IgY) is extremely low.For preparation, cleaning and detection of Ig Y it would beof advantage to have a "Protein Y" available analogous toprotein A and protein G for mammalian antibodies. Untilnow, the search for .Protein?" was unsuccessful.IgY has been usedfor routine diagnostic work covering thefollowing subjects:J. Identification of the host species from abdominal blood ofhaematophageous insects;2. IgY-anti-horse-Ig-PO conjugate for ELISA on dourine;3. FITC-conjugated Ig Y-antirabies for diagnostic work onrabies;4. FITC-conjugated Ig Y against avian virus diseases(Newcastle dis., Infectious bronchitis, Gumboro). In allcases satisfactory results have been achieved.

Keywords: IgY, commercial availability, "Protein Y",routine diagnostic work

1 Introduction

The Laboratory for Immunology andDiagnostics at the BgVV which isaccommodating the project on Igy isconcerned with development, produc-tion and distribution of reagents for thediagnosis of zoonoses and animaldiseases. Additionally, a second project- the BgVV/GTZ sponsored Service-Laboratory - is attached to the Labora-tory for Immunology and Diagnostics.Within the Service-Laboratory, serolo-gical techniques are adapted accordingto poor financial conditions of de-veloping countries by selfproduction ofserological reagents, cleaning and re-use of plastic material, a.s.o.The IgY project which developed

uncomplicated techniques for immuni-sation of hens, isolation of egg yolkantibodies, and their applicability invarious test systems creates continuousconsiderations in regard of changingfrom mammalian derived antibodies toegg yolk antibodies for both, the Natio-nal Laboratory for Immunology and

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Zusammenfassung: Dotterantikorper und ihr Einsat; in derRoutinediagnostik.Die kommerzielle Yerfugbarkeit von Dotterantikorpern(Ig Y) ist entsprechend den Angaben aus Linscott's Directoryof Immunological and Biological Reagents (1994/95) zurZeit noch als ausgesprochen gering anzusehen.Fur die Prtiparation, Reinigung und Markierung von Ig Yware es vorteilhaft, ein "Protein Y" - analog dem Protein Aund Protein G for mammdre Antikorper - verfiigbar zuhaben. Die Suche nach einem "Protein Y" fiihrte bishernicht zum Erfolg.Untersuchungen mit avidren Antikorpern wurden auffolgenden Gebieten durchgefiihrt:1. Identifirierung der Wirtstierart aus dem Abdominalbluthdmatophager Insekten;2. IgY-anti Pferd Ig-Prr-Konjugat fur den Einsatz in derBeschalseuchediagnostik;3. lg Y-anti- Tollwut-Fl'I'CsKonjugatfur die Tollwutdiagno-se;4. IgY-anti-avidre Virusinfektionen (Newcastle Disease, In!Bronchitis, Gumboro) konjugiert mit FITe. Mit den vorge-stellten Priiparationen wurden gute Ergebnisse erzieli.

Diagnostics and the internationally ori-entated Service Laboratory.Regarding commercially available

polyclonal antibodies being producedworldwide, these antibodies are listedin Linscott's Directory of Immunologi-cal and Biological Reagents (1994/95).In this directory, not only antigenicspecificities are quoted but as well thedonor animal species. Table 1 providesthe number of polyclonal antibodiesfrom chicken in relation to the totalnumber of polyclonal antibodies recor-ded.

297 from almost 16.000 is less than2% of polyclonal antibodies derivingfrom chicken, and only three fromthese have been prepared from eggyolk, the rest apparently from chickensera. Nevertheless, chicken as donoranimals occur in every group and itmay be concluded that in general,chicken are potent antibody producers.Obviously, the IgY-technology needsfurther propagation.

Table 1: Linscott's Directory, polyclonal antibodies. Number of chicken derivedantibodies from the total of polyclonal antibodies

Antibody Group Derived fro ChickenTotal in Group

anti-Immunoglobulinanti-C-Fractionsanti-Cells& Tissue Proteinsanti-Miscellaneousanti-Infectious Agents

Total

6261441

295044551892

13724754318

15999 297

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_ST_A_A_K ~~~---

~c~..~.Three subjects are presented:

1. Search for "Protein Y",2. .Bloodmeal identification",3. IgY for routine diagnostic work.

2 Search for "Protein Y"

Protein A from a specific Staphylococ-cus aureus strain and protein G fromgroup G streptococci are heat stablecell wall proteins having the propertyof specific binding to the Fe portion ofa wide range of immnunoglobulins(table 2).Neither chicken IgG nor IgY are

specifically bound by protein A or G.For affinity chromatography and formarker techniques, protein A and Ghas gained a very high importance forisolation, concentration and labellingof IgG isotypes from mammalian sera,and it can be expected that the samewould be true for "protein Y" whichremains still to be detected.While searching for such a protein,

1000 staphylococcus and streptococcusstrains have been screened by a specifi-caJly designed technique but till nowwithout success.

3 Bloodmeal identification

In Africa, tsetse flies transmit trypano-soma infections causing sleeping sick-ness in humans and nagana in animals.It is estimated that roughly 10 Mio km2

are inhabited by tsetse flies and theannual losses by death, emaciation,loss of meat, milk, and incapability towork as draught animals are calculatedto amount to 5 billion US$ per annum.Additionally, trypanosoma infectionsmay lead to immunosuppressioncausing secondary, mainly bacterialinfections to flare up.Good knowledge regarding the beha-

viour of tsetse flies are a prerequisitefor successful campaigns against theseparasitic insects. Tsetse flies have hostpreferences for certain animal speciesfrom which they take their bloodmeal.The knowledge about these preferencesin given areas in connection with sea-sonal behaviour provides means forspecific campaigns against tsetse flies.Host preferences can be detected by

analysing abdominal blood from flies

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Table 2: Comparison of species andIg-isotype reactivity of protein A and G

Immunoglobulin Protein A Protein G

Human 19G1 ++ ++IgG2 ++ ++IgG3 ++IgG4 ++ ++

Mouse 19G1 +/- +/-IgG2a ++ ++IgG2b ++ ++IgG3 ++ ++

Rat IgG1 +/- +/-IgG2a ++IgG2b +/-IgG2c ++ ++

Rabbit IgG ++ ++

Bovine IgG1 ++IgG2 ++ ++

Sheep IgG1 ++IgG2 ++ ++

Goat IgG1 +/- ++IgG2 ++ ++

Horse IgG ++

Chicken IgG/lgY

for identification of the animal speciesfrom which this blood has been taken.The procedure includes an ELISA sy-stem with various anti-animal speciesantisera against which abdominalblood samples dried onto filter paperare tested after reconstitution. Typicalreaction patterns of control antigenstested against homologous and hetero-logous antisera from rabbits are presen-ted in table 3.

If closely related species are to beidentified, specific antisera have to beabsorbed with antigen (serum) fromcompeting species and the result ofsuch absorption for the differentiationof domestic pig, warthog and bushpigis demonstrated in table 4.When transferring the production of

antibodies from rabbits to chicken itwas observed that high titred IgY anti-animal species antibodies could beisolated from egg yolk of immunisedhens. The principle of absorption withcompeting antigens was successfullyapplied though the phylogenetical di-stance from chicken to mammalianspecies seemed to require a more inten-se absorption procedure (Graph I and2).The bloodmeal identification service

has been transferred to Burkina Paso,and it is planned to use IgY and not anymore rabbit sera as a source for antibo-dies.

4 IgY for routine diagnostic work

The test prescribed by O.I.E. for sero-logical diagnosis of dourine (Trypano-soma equiperdum) in solipeds is thecomplement-fixation test (eFT). Thedevelopment of an ELISA test becamepossible by acquiring larger quantitiesof serum from infected horses. Theconjugate used for this ELISA test isIgYvanti-horse Ig conjugated to POD.An application for the acceptance ofthis test as a prescribed or alternativetest for international trade was directedto the O.I.E. A further advantage ofELISA in the sense of animal protec-tion is a comparatively low consumpti-on of antigen which is produced from

Table 3: Bloodmeal identification: Control set 1

Antigen Antisera against

2 3 4 5 6 7 81 Dog ++2 Elephant +++3 Cat ++4 Chicken "'""'""'"S Hippopotamus- +++6 Crocodile +++7 Rat +++8 Lion + +++

Cross-reaction between lion (8) and cat (3) indicate the relationshipof felidae

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Table 4: Bloodmeal identification: Control set 2

Antigen Antisera against2 3 4 5 6 7

1 Human +++2 Ruminant +++3 Monitor lizz.- +++4 Suidae ++ ++ ++ ++5 Domest.pig- ++ ++6 Warthog +++ ++7 Bushpig +++ ++8 Avian

Anti-Suidae serum (4) non-absorbed; anti-domest.pig (5), anti-warthog-(6), anti-bushpig-(7) sera absorbed withcross-reacting antigens to produce specific reactions.

Titersteps (1:50 ' 1)7,--------------,-------6

5

4

8

Specific pathogen free (SPF) birds arenot required in this context since affini-ty chromatography with appropriateligands permits the isolation of specificantibodies out of a pool of specific andnon-specific antibodies. Affinity chro-matography has been adapted to ahome-made system by using pulveri-zed polystyrene material (microtitreplates) as a matrix material (Staak etaI.,1996).

++

Titersteps (10200 • 1)8,-------------------7

654321o _1- ---'--_

I ~

Original reaction 1st Absorption 2nd Absorption

Homologous and heterologous reactions

Original reaction After 4 absorptions

Homologous and heterologous reactions

_ Dog

~ Horse

I '':::1 Suidae

G!.l:J Lion

~~ Human

o Hippopot.

Antig.for absorpt.:1.Bov ine.z.Liori/H ippo

~ Bovine

_ Warthog

~ Pool 1

Pool 1: Rodent, hippopot., elephant, lionPool 2: Rabbit, dog, horse

~ Dom.pig

D Pool 2

CJ Bushpig

G1 Human

m Bovine

Figure 1: IgY-antidog: Original reaction and reaction after 1.und 2. absorption

artificially infected rats: The amount ofantigen needed for conducting ELISAis 5% of the amount to be used incomplement-fixation test.For the identification of rabies in

brain smears from suspected cases,fluorescent anti-rabies conjugates arefrequently used. Commercially availa-ble conjugates are normally derivedfrom rabbit sera.Chicken vaccinated repeatedly with

rabies vaccine (Rabipur, Behringwer-ke, Marburg) responded well with highIgY -anti-rabies antibodies which couldbe conjugated to FITC by the sameprocedure as used in the FITC-conju-gation of mammalian antibodies. Re-sulting specific fluorescence was of thesame quality as found in mammalianderived preparations.IgY -FITC conjugates directed

against avian virus diseases have beenprepared as ajoint project of the JordanCentre for Veterinary Vaccines in Am-man and the Service Laboratory atta-

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Figure 2: IgY-antiwarthog: Original reaction and final reactionafter 4 absorptions

ched to the Laboratory for Immunolo-gy and Diagnostics at the BgVV. Spe-cific conjugates were directed againstNewcastle disease (ND), infectiousbronchitis (IE) and gumboro (IEDV).These conjugates were to be used inbirds suspected to have died of one ofthe diseases mentioned by direct fluo-rescent staining of impression smearsfrom affected organs. In artificiallyinfected chicken, virus antigen couldbe specifically detected in all organsexamined. High virus quantities weredetected in the spleen, trachea andlungs following infection with NDV,in the trachea following infection withlEV and in the bursa of Fabriciusfollowing the infection with IEDV(Gervelmeyer, 1995).The IgY technology seems to be well

suited to meet many of the diagnosticrequirements in European and de-veloping countries. One of the advan-tages for developing countries is theavailability of chicken everywhere.

ReferencesGervelmeyer, A. (1995). Herstellung van

virusspezifischeti vitellinen Antikorpernund deren Einsat; im dlrekten Fluores-zennest zum Nachweis der Erreger derGefliigelkrankheiten Newcastle Disease,Infektiose Bronchitis und CumboroDisease. FU Berlin: Vet. Med. Diss.,J.Nr. 1835.

Linscott's Directory of Immunological andBiological Reagents, Eighth Edit. (1994195). Santa Rosa Calif., USA: W.D.Linscott.

Staak C., Salchow, F., Clausen, P.-H. undLuge, E. (1996). Polystyrene as an Affi-nity Matrix for the Purification of Anti-bodies. 1. Immunol. Meth., in press.

Correspondence addressDr. Christian StaakBundesinstitut fur gesundheitlichenVerbraucherschutz und Veterinar-medizin BgVVBereich MarienfeldeDiedersdorfer Weg 1D-12277 Berlin

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