The Production of Avian (Egg Yolk) Antibodies: IgY

ATLA

24, 925-934, 1996

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The Production of Avian (Egg Yolk) Antibodies: IgY The Report and Recommendations of ECVAM Workshop 21 1,2

Riidiger Schade,3 Christian Staak,4 Coenraad Hendriksen,5 Michael Erhard,6 Herbert Hugl,7 Guus Koch,s Anders Larsson,9 Wolfgang Polimann,lO Marc van Regenmortel,l1 Eric Rijke,12 Horst Spieimann,I3 Harry Steinbusch I4 and Donald Straughan I5 3Institut fur Pharmakologie und Toxikologie, Universitiitsklinikum Charite, Dorotheenstrasse 94,10117 Berlin, Germany; 4BgW, Diedersdorfer Weg 1,12277 Berlin, Germany; 5National Institute of Public Health and Environmental Protection (RIVM), Antonie van Leeuwenhoeklaan 9, 3720 BA Billhoven, The Netherlands; 6Institut fur Physiologie, Tierarztliche Fakultat, Ludwig-Maximilians Universitcit, Veteriniirztstrasse 13, 80539 Munchen, Germany; 7Bayer AG, ZF-FDM, Rheinuferstrasse 7-9, 47829 KrefeldUerdingen, Germany; 8Department of Avian Virology, ID-DLO, 8200 AB Lelystad, The Netherlands; 9Department of Clinical Chemistry, University of Uppsala, 75 123 Uppsala, Sweden; lOInstilute of Microbiology, Johann Gutenberg University, 55122 Mainz, Germany; 11 Institut de Biologie Moleculaire et Cellulaire du CNRS, 67084 Strasbourg, France; 12Intervet International BV, Wim de Korverstraat 35, 5830 AA Boxmeer, The Netherlands; 13ZEBET, BgW, Diedersdorfer Weg 1, 12277 Berlin, Germany; J4Department of Psychiatry and Neuropsychology, Rijksuniversiteit Limburg, 6200 MD Maastricht, The Netherlands; 15FRAME, Russell & Burch House, 96-98 North Sherwood Street, Nottingham NG1 4EE, UK

Preface This is the report of the twenty-first of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM), ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become

well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of ifl. vitro tests and their potential uses, and make recommendations about the best ways forward (1).

The workshop on The Production of Avian Antibodies was held in Berlin, Germany, on

Address for correspondence: Dr C. Hendriksen, RIVM, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthouen, The Netherlands. Address for reprints: ECVAM, TP 580, JRC Environment Instilute, 21020 Ispra (VA), Italy. 'ECVAM - European Centre for the Validation of Alternative Methods. 2This document represents the agreed report of the participants as individual scientists.

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22-24 March 1996, under the chairmanship of Christian Staak (BgVV, Berlin, Germany). The participants at the workshop, who were representatives from academia, industry and government, reviewed the current status of immunoglobulin (Ig) Y production in the chicken. Particular emphasis was placed on discussion of: a) the housing of chickens; b) immunisation protocols; c) egg-laying behav-

iour; d) the isolation and purification of IgY; and e) the successful replacement of the use of mammalian antibodies by IgY. The main issues discussed and recommendations made in connection with these topics are summarised in this report.

R. Schade et al.

The main type of Ig isolated from egg yolk is generally referred to as "IgY"j other Ig classes are present, but only in negligible amounts. Structurally, IgY is identical to the major Ig found in serum, but it is different from mammalian IgG (Figure 1). There is still controversy about the relative concentrations of the different types of Ig found in egg yolk and serum; the data available indicate that IgY is more concentrated in yolk than it is in serum (4).

Figure 1: Structures of mammalian IgG and avian IgY

Introduction Hinge

In addition to their therapeutic importance in medicine, monoclonal antibodies and poly-

clonal antibodies are of great value in biological research, where they serve as essential components in a variety of diagnostic systems used for the qualitative and quantitative determination of a wide range of substances. It is not surprising, therefore, that the growing interest in alternative methods has focused not only on the quality control of antibodies, which was covered in a previous ECVAM workshop (2), but also on the methods used for the production of antibodies, as discussed at this workshop. Antibody production normally requires the use of laboratory animals (mainly rabbits, but also mice, rats and guinea-pigs) or larger mammals, such as horses, sheep and goats. The procedure involves two steps, each of which causes distress to the animals involved: a) the immunisation itself; and b) bleeding, which is a prerequisite for antibody preparation. The use of chickens for antibody production, as opposed to mammals, represents both a refinement and a reduction in animal use. It is a refinement in that, the second painful step, the collection of blood, is replaced by antibody extraction from egg yolk. It entails a reduction in the number of animals used because chickens produce larger amounts of antibodies than laboratory rodents. In fact, it has been known for over a hundred years that the immunisation of a chicken induces the production of similar concentrations of specific antibodies in both egg yolk and serum (3).

/gG

I Cy2

Cy3

/gY

Cu3 Cu4

Despite the advantages associated with producing antibodies in chickens (5; Table n, the method is not as widely used as might be expected. This could be due to several factors, such as incorrect information about the procedure, a lack of appropriate experience with the technique, or simply a reluctance to use new methods. In the opinion of the workshop participants, the wider use of egg yolk antibodies is being hampered by problems with keeping the chickens and with antibody extraction.

ECVAM Workshop 21: avian antibodies

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Table I: Comparison of the characteristics of mammalian IgG and avian IgY

Mammalian IgG

Avian IgY

Antibody sampling

invasive

non-invasive

Antibody yield

200mg IgG per bleed (40ml blood)

50-100mg IgY per egg (5-7 eggs per week)

Antibody yield per month

200mg

- 1500mg

Specific antibody yield

-5%

2-10%

Protein Nprotein G binding

yes

no

Interference with mammalian IgG

yes

no

Interference with rheumatoid factor

yes

no

Activation of mammalian complement

yes

no

Based on Schade et al. (5).

The Chicken as a Laboratory Animal A basic requirement for the use of chickens is the availability of housing conditions which favour species-specific behaviour. A suitable cage for chicken-specific housing must, at the very least, conform to the minimum standards outlined in Table 13 of Directive 8616091EEC (6) and in the Euro· pean Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (7). Wherever pas· sible, it is desirable that larger cages than those described are used. A specific, commercially available, cage for the housing of two chickens (128 x 65 x 80cm) is recommended by the Swiss veterinary administration (Schweizer Bundesamt fur Veterinarwesen) . Housing chickens in cages, under laboratory conditions is advantageous, in that the chickens can be readily located and their health can be easily monitored. Although the housing of chickens in social groups on the floor is desirable from the perspective of animal welfare/social behaviour, it is coupled with the problem of egg identification (trap· ping nests require more individual care), and with an increased risk of infection (especially for chickens housed outside). By keeping a brown and a white hen together in one cage, the eggs can be identified unequivocally.

Antibodies can be produced by using chickens bred for commercial egg production as well as those which have been bred free from specific pathogens (SPF chickens I. It is preferable to use chickens used for breeding purposes than those used for egg production, because the health status of breeding animals is often better controlled. When out· bred chickens are used, it should be borne in mind that 10-15% of them may be non· responders or low-responders to a certain antigen. Some inbred chicken strains have been shown to be low-responders to certain antigens, such as Salnwllella pullonlln bacterin, human serum albumin, and synthetic peptides; nevertheless, strains B14, B17 and B21 show good antibody responses against these ,proteins. SPF chickens can be obtained from some commercial suppliers in Europe (for example, F.E. Lohmann, Cuxhaven, Germany) and in the United States (for example, Spafas, Preston, CT, USA). Adult SPF chick· ens are relatively difficult to obtain, and therefore usually have to be raised in the laboratory. Eggs from genetically defined flocks can be obtained from several sources (8). Commercial laying chickens are not only cheaper to purchase, but they can also be obtained just before they come into production, thereby further reducing the costs associated with antibody production. The

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R. Schade et al.

advantage of using SPF chickens over egg~ laying chickens is that the former generally give higher antibody titres, although conflicting findings have been reported (9), In addition, several viral diseases may cause transient immunomodulatory effects which can interfere with antibody production. Thus, if the antibodies produced are to be used for therapeutic purposes, the use of SPF chickens is compulsory. Another important consideration is the egg-laying capacity of the chicken, and the possible factors which may affect it. One such factor could be immunisation using Freund's complete adjuvant (PCA), or the antigen itself. According to some reports, FCA does not influence egg production as much as the antigen itself, as has been shown, for example, for substances from Ascaris sltum (10); the results of this study indicated that egg-laying capacity is influenced primarily by events other than immunisation.

Recommendations 1. Chickens should be kept under conditions

which encourage natural behaviour. Research on environmental enrichment should be supported, so that housing conditions can be further improved. 2. It is preferable to keep chickens indoors, and to restrict entrance to chicken houses to authorised personnel only. Such personnel should have no contact with commercially maintained poultry.

3. For scientific purposes (laboratory work), conventional housing (cages, with groups of at least two hens) should be used. 4. When antibodies are to be used for therapeutic purposes, the use of SPF chickens is compulsory. 5. Although chicken strains used commercially for egg production give an acceptable antibody response, it is preferable to use inbred strains in order to induce higher antibody responses. 6. It is preferable to immunise chickens before they begin to produce eggs, because the stress induced by handling them can have an adverse effect on egg production, as can the nature of the antigsn or adjuvant used.

Chicken Immunisation Protocols Various protocols for raising antibodies in chickens and extracting them from eggs have been reported, but these are difficult to compare with respect to the type and dose of antigen used, and the route and frequency of injection. The discussion of chicken immunisation protocols during the workshop resulted in agreement upon a number of recommendations (summarised in Table 11).

Adjuvant The type and quality of the adjuvant used are of critical importance in determining the

Table II: Recommendations relating to chicken immunisation protocols

Recommendation Adjuvant

Freund's incomplete adjuvant, Specol, lipopeptide (Pam3-Cys-Ser-[Lys]4; 250/Lg)

Antigen dose

10ng-1mg (preferably 10-100/Lg)

Injection site

Intramuscular (field studies; young laboratory chickens) Subcutaneous (older laboratory chickens)

Injection volume

< 1ml

Injection frequency

2-3 times; boosters during laying period

Vaccination interval

4-8 weeks

Use of chickens

Entire laying period (about 1 year)

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immune response, which should, ideally, be the induction of high serum and egg yolk antibody titres. The use of an adjuvant, especially FCA, can lead to a local tissue reaction at the injection site (11). In general, the expected antibody response can be generated by using an oil-emulsion type of adjuvant, such as Freund's incomplete adjuvant (FIA). No differences have been seen in the IgY response when FIA has been used for the primary immunisation instead of FeA. Other types of adjuvant can also be used, such as Specol (12; product no. 792500, ID-DLO, Lelystad, The Netherlands) and the lipopeptide, Pam3-Cys-Ser-(Lys)4 (M.H. Erhard et oZ., submitted for publication). The adjuvants AlP04, Al(OH)3 and saponin have been found to induce only very low antibody responses. Thus, it is important to first test the efficacy and quality of emulsion-type adjuvants according to standardised procedures (13).

Antigen dose Initially, various concentrations of the antigen should be combined with the adjuvant, since the immune response is influenced by the type of antigen. Vaccination volume It is usual to vaccinate chickens that are at least 7 weeks of age, preferably at two injec-

tion sites, with volumes ofO.5-1ml. The total volume injected will affect the tissue reaction induced. Route of injection

For practical and economic reasons, chickens kept under field conditions are vaccinated intramuscularly in the breast muscle. In the laboratory, chickens can also be vaccinated subcutaneously in the neck. With very young animals, it may be preferable to inject intramuscularly into the breast muscle, because subcutaneous injections are more difficult to perform and can therefore cause more distress. Intramuscular injection in the leg should be avoided, since it can lead to lameness. Vaccination frequency

The total number of vaccinations required will depend upon the type and dose of the antigen, as well as on the particular adjuvant

employed. In any case, at least two immunisations should be given. If the antibody titres begin to decrease, booster immunisations can be·given during the laying period, Vaccination interval

A primary vaccination and a booster should be given before the laying period, with an interval between them of at least 6 weeks for emulsion-type adjuvants (13) and 4 weeks for lipopeptide adjuvants. Yolk antibody titres should be checked 14 days after the last immunisation; if the antibody titres are low, revaccination should be considered. Length of time for which chickens can be used

In principle, chickens can be used for the whole of the laying period, depending on the antibody titres induced. It is advisable to start with a group of chickens, and to select high responding animals which can then be kept for a longer period of time.

Nomenclature Historically, the low molecular weight (MW) Ig found in avian serum was known as IgG, by analogy with its mammalian counterpart. It has become clear, however, that this is inappropriate due to the fundamental structural differences between IgG and IgY (Figure 1). In fact, no IgG-like antibody with a heavy y chain of 50,000 Da has been found in the chicken (14). The term IgY was originally coined to refer to the larger MW Ig found in egg yolk, but it is now accepted that IgY is the major antibody in both blood and yolk. The heavy (y) chain of IgG consists of four domains: the variable domain (VH) and three constant domains (Cyl-Cy3). The Cyl domain is separated from Cy2 by a hinge region, which gives considerable flexibility to the Fab fragments. In contrast, the heavy chain ofIgY (u) has an MW of 65,000 Da, does not have a hinge region, and possesses four constant domains (CuI-Coo) in addition to the variable domain (Figure 1). Sequence comparisons between IgG and IgY have shown that the Cu2 and Cu3 domains of IgG are closely related to the Cu3 and Coo domains, respectively, of IgY, while the equivalent of the Cu2 domain is absent in the y chain, having been replaced by the hinge region (14).

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Sequence data support a phylogenetic tree in which IgY gave rise to both IgG and IgE. Sequence comparisons of the D, y and € chains show that they are more closely related to each other than is anyone of them to either ~ (lgM) or " (lgA). In fact, IgY is more closely related to IgE than to IgG. The close similarity of IgY to IgE is apparent from the number and organisation of the intradomain and intrachain disulphide bonds. In conclusion, the term "IgG" should not be used for chicken antibodies, since it does not conform to our current knowledge on antibody structure.

Isolation and Purification Methods Several methods can be used for the extrac~ tion of IgY from egg yolk, and commercial extraction kits are available (15). One of the most frequently used procedures involves protein precipitation with ammonium sulphate, dextran sulphate or polyethyleneglycol (PEG); separation by ion exchange chromatography is also used. There is, in fact, a surplus of effective extraction methods (16), which could be problematic in that potential users of IgY technology may have no rational basis for choosing one method rather than another. In practice, the choice of a specific extraction procedure is usually influenced by the intended application of the antibody, as well as by the experience of the laboratory concerned (17). A particularly efficient method consists of two successive precipitations in PEG, by first using 3.5% PEG to remove fatty substances, and then 12% PEG to precipitate the IgY. An improvement of this method incorporates an emulsification step, adding one volume of chloroform to one volume of egg yolk, rather thim using the 3.5% PEG precipitation step (18,19). Despite the published evidence, there was no agreement by the workshop participants as to which method, or methods, should be recommended for general use. If possible, such an agreement should be reached, in order to improve the general acceptance and wider use of avian antibodies.

Recommendations 1. A detailed and careful comparison of the different methods for purifying IgY

R. Schade et al.

should be undertaken. Rudiger Schade (Universitatsklinikum Charite, Berlin, Germany) and Anders Larsson (University of Uppsala, Sweden) have agreed to undertake this with two aliquots of IgY supplied by Christian Staak (BgVV, Berlin, Germany), in accordance with detailed protocols which will be supplied by other participants at the workshop. It is generally assumed that about 100mg of IgY can be recovered per egg yolk. 2. A standard method should be established for determining IgY concentration. An attempt will be made to develop such a method by using monoclonal antibodies to IgY supplied by Guus Koch (ID-DLO, Lelystad, The Netherlands) and Michael Erhard (Ludwig-Maximilians Universitat, Munich, Germany). The amount of active antibody in the different purified IgY preparations, and the affinity of the antibody, will then be measured by Marc van Regenmortel (lnstitut de Biologie Moleculaire et Cellulaire du CNRS, Strasbourg, France), by using biosensor technology (BIAcore™). The key issues on which further information needs to be sought are: a) percentage recovery; b) purity, as assessed by electrophoresis; c) percentage of active antibody in the total IgY fraction (as measured by ELISA) compared with the total protein concentration; d) the cost of the materials; and e) the amount of time required for IgY preparation and the associated labour costs.

Specificities of Avian Antibodies from a Phylogenetic Point of View The immune response in an antibody-producing animal tends to increase as its phylo-

genetic difference with the animal used as the antigen source increases. Thus, chicken antibodies recognise more epitopes on a mammalian protein than the corresponding rabbit antibody does, making it advantageous to use IgY in immunological assays of mammalian proteins. This is especially true when the antigen is a highly conserved protein, such as a hormone (20-22). Moreover, if a secondary antibody of mammalian origin is used, the phylogenetic difference will result in a further amplification, since three to five times more of the secondary antibody will

ECVA11 Workshop 21: avian antibodies

bind to chicken IgY than occurs with rabbit IgG (23). Another advantage of chicken IgY over mammalian antibodies is that it does not activate the complement system; the latter has been shown to reduce antigen binding and cause false negative results (24). Chicken IgY does not react with anti-mammalian antibodies in human serum, such as rheumatoid factors and human anti-IgG. In immunological assays, the interference caused by these antibodies can be problematic (25), particularly as the sensitivity of the assay increases. Thus, if chicken IgY is used, interference by anti-mammalian IgG antibodies is eliminated (26), Chicken IgY does not bind to human or bacterial Fc-receptors, such as Staphylococcal protein A or Streptococcal protein G (4). Thus, IgY can be used for microbiological assays without the risk of interference by Fc-receptors. Nevertheless, it is a disadvantage that neither protein A nor protein G can be used for the purification of chicken IgY (26-28). The activation of Fc-receptors by the antigen-antibody complex results in a change in the surface proteins, which may interfere with the assay (29).

The structure of IgY is more rigid than that of rabbit antibodies, and it is less efficient in precipitating antigens. Thus, to achieve the same result as with a rabbit antibody, a larger amount of IgY will be needed; this may increase the cost of the assay (14). Recommendations

t. The use of chicken IgY offers several advantages over mammalian antibodies. To increase the use of IgY, techniques for both direct and indirect labelling must be optimised. 2. There is a growing need for secondary antibodies to IgY, labelled with different markers (enzymes, fluorescent markers), to be made available commercially (such as the monoclonal antibodies to chicken IgY, available from Connex [Munich, Germany] and ID-DLO [30, 31]).

Successful Substitution of Mammalian Antibodies with Egg Antibodies Commercially available polyclonal antibodies which are produced worldwide are listed

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in Linscott's Directory of Immunological and Biological Reagents (32). The donor animal species as well as the antigenic specifications are quoted; the number of polyclonal antibodies raised in chickens, relative to the total number of polyclonal antibodies listed, is less than 2%. Nevertheless, chickens are given as donor animals in every antibody group. Thus, it may be concluded that, in general, chickens are potent antibody producers, and their immunological responsiveness is similar to that of mammals. A few examples to illustrate this point were presented during the workshop: a) antibodies against human troponin I have been produced for use as a reagent in a fully automated immunodiagnostic system; b) IgY anti-horse Ig conjugated to peroxidase has been incorporated into a test kit for the diagnosis of dourine (Trypanosoma equiperdum; 33); c) IgY anti-Newcastle disease, anti-infectious bronchitis and anti-Gumboro have been conjugated to fluorescein isothiocyanate, enabling the detection of these avian viral diseases by using a fluorescent antibody technique (34); and d) IgY directed against blood components from several mammalian species is as capable of differentiating between these species as rabbit antibodies are (35). Apart from the advantages associated with the use of IgY antibodies, there are some potential disadvantages. One of these, the failure of IgY to bind to protein A and protein G, precludes the use of these proteins in IgY isolation. The workshop participants felt that this was not a major problem, since various simple procedures for IgY extraction already exist. In addition, the search for a "protein Nprotein Glike" IgY isolation procedure is currently being supported by a grant from the Bundesministerium fur Bildung und Forschung in Germany. Another potential disadvantage of IgY is that it is less able to precipitate antigen compared with IgG. Better precipitation can sometimes be achieved by increasing the salt concentration in the buffer solution, but the poorer precipitation of antigen by IgY remains a shortcoming which may limit its use in automated diagnostic systems. Various other criticisms of the use of IgY are mostly subjective in nature, and are principally due to a lack of proper information on the topic.

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Summary of Conclusions and Recommendations The workshop participants agreed that more-widespread use of chicken IgY should be promoted, since this method satisfies scientific and commercial interests as well as a concern for animal welfare (Figure 2). However, it must be stressed that there is no justification for completely substituting the use of mammalian polyclonal antibodies with avian antibodies, since IgY is immunologically distinct from IgG, and is therefore neither better nor worse. Thus, diagnostic tools could be improved by exploiting the advantages of IgY in combination with IgG. In addition, the use of IgY will sometimes offer new approaches for dealing with scientific problems in immunology. Before deciding on the method to be used for antibody production, animal welfare considerations should he taken into account; the immunisation of a chicken instead of a mammal may often be an appropriate approach. In summary, there are many arguments supporting the wider use of avian antibod~ ies. It is hoped that the outcome of this ECVAM workshop, and the continuing activities of the individual participants, will stimulate greater interest in this field and that the report will act as a source of information to potential users of IgY technology. The main recommendations pertaining to the use of avian antibodies are summarised below:

R. Schade et ai.

Figure 2: Advantages of IgY technology

REFINE

IgY Technology

1. Chickens should be kept under conditions which encourage their natural behaviour. Research on environmental enrichment should be supported, so that housing conditions can be further improved. 2. It is preferable to keep chickens indoors, and to restrict entrance to chicken houses to authorised personnel only. Such per~ sonnel should have no contact with com~ mercially maintained poultry. 3. For scientific purposes (laboratory work), conventional housing (cages, with groups of at least two hens) should be used. 4. When antibodies are to be used for therapeutic purposes, the use of SPF chickens is compulsory. 5. Although chicken strains used commercially for egg production give an accept~ able antibody response, it is preferable to

ECONOMICS

use inbred strains in order to induce higher antibody responses. 6. It is preferable to immunise chickens before they begin to produce eggs, because the stress induced by handling them can have an adverse effect on egg production, as can the nature of the anti~ gen or adjuvant used.


7. With respect to protocols for chicken immunisation, recommendations about the adjuvant, antigen dose, injection site, volume and frequency, vaccination inter~ val, and the period for which chickens should be used are summarised in Table II. 8. The term "IgG" should not be used for chicken antibodies, since it does not con~ form to our current knowledge on anti~ body structure.

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8.

9.

10.

9. A detailed and careful comparison of the different methods for purifying IgY should be undertaken. 10. A standard method should be established for determining IgY concentration. 11. Techniques for both direct and indirect labelling of IgY need to be optimised, and there is a growing nee