WO2006117007A1 - Methodes d'obtention d'anticorps specifiques de l'antigene - Google Patents

Methodes d'obtention d'anticorps specifiques de l'antigene Download PDF

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Publication number
WO2006117007A1
WO2006117007A1 PCT/DK2006/050017 DK2006050017W WO2006117007A1 WO 2006117007 A1 WO2006117007 A1 WO 2006117007A1 DK 2006050017 W DK2006050017 W DK 2006050017W WO 2006117007 A1 WO2006117007 A1 WO 2006117007A1
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Prior art keywords
antibodies
antigen
peptides
animal
peptide
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English (en)
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Janus Beierholm Larsen
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ANTIBODY PROJECT APS
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ANTIBODY PROJECT APS
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Publication of WO2006117007A1 publication Critical patent/WO2006117007A1/fr
Priority to DK07722590.2T priority Critical patent/DK2027158T3/da
Priority to PL07722590T priority patent/PL2027158T3/pl
Priority to PCT/DK2007/000211 priority patent/WO2007124755A1/fr
Priority to EP11188731A priority patent/EP2463306A1/fr
Priority to EP07722590A priority patent/EP2027158B1/fr
Priority to US12/299,247 priority patent/US8859233B2/en
Anticipated expiration legal-status Critical
Priority to DKBA201100184U priority patent/DK201100184Y4/da
Priority to US14/493,468 priority patent/US9914766B2/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • the present invention relates to the field of antibodies.
  • the invention concerns methods for obtaining polyclonal antibodies from avian species and more specifically from chicken (Gallus gallus domestica).
  • the present invention also relates to methods for obtaining antigen specific antibodies.
  • Antibodies are widely used as therapeutic agents, diagnostic tools, research tools, etc.
  • Polyclonal antibodies are conventionally obtained by immunising a suitable host, usually mammalian with a composition comprising the molecule of interest as an antigen and a suitable adjuvant. Several weeks later, polyclonal antibodies specific to the antigen can be harvested by bleeding the animal and collecting the serum.
  • polyclonal antibodies bind different parts of the antigen and individual polyclonal antibodies might likewise bind virtually the same parts of an antigen with different affinities.
  • Polyclonal antibodies are usually obtained from mammals such as rats, mice, goats, horses, sheep, rabbits, etc. A large amount of experimental animals are used to generate different antibodies - one animal is used per antigen. It follows that the animal will experience discomfort associated with the immunisation and in particular the bleeding procedures. It also follows that it is expensive and laborious to obtain antibodies specific to a large number of different antigens since a similar large amount of animals is required.
  • mice were immunised with 10 ⁇ g of 5-10 different protein antigens having sizes ranging from about 12 to about 48 kDa in order to solve the bottleneck problem in providing monoclonal antibodies to proteins encoded by the human genome.
  • It is also possible that increasing the number of immunogens for each animal may ultimately lower the number of positive clones in any one fusion.
  • - chicken antibodies are acid and heat resistant and might therefore be orally applied to prevent or to cure intestinal diseases of young animals or humans.
  • mice effect of subcutaneous and oral sensitization with untreated bovine milk and homogenized bovine mik, In Vivo, 3, 271, 1989.
  • the present invention aims at solving these problems by providing a method of obtaining polyclonal antibodies specific to a pool of antigens, wherein the method comprises immunising a vertebrate animal with a pool of antigens, and subsequently collecting polyclonal antibodies.
  • Antibodies have been raised in animals for decades - using one antigen per animal. According to the present invention however, antigen specific antibodies can surprisingly be obtained by immunising an animal with a large number of antigens. It might thus be concluded that a technical prejudice have been overcome by successfully immunising animals with a large number of antigens.
  • the invention likewise relates to a method of isolating antigen specific antibodies from an antibody pool, wherein the method comprises immobilising the antigen, contacting the antibody pool with the immobilized antigen, removing unbound antibody, and recovering antigen specific antibodies.
  • affinity chromatography is used for capturing antigen by means of immobilised antibodies.
  • affinity chromatography is used for capturing antigen by means of immobilised antibodies.
  • the present invention relates to the products resulting from the various methods.
  • the present invention relates to a method of obtaining polyclonal antibodies comprising antigen specific antibodies, wherein said method comprises the following steps:
  • composition comprising: (i) about 15 or more different peptide antigens, said peptides being conjugated with a carrier and having a length of at least 6 amino acids, and (ii) an adjuvant,
  • the principle of immunising an animal, preferably a bird of the gallinacean type, and most preferably of the species Gallus gallus domestica with a large amount of different antigens is a principle that provides tools for obtaining polyclonal antibodies in a much more fast and inexpensive way than previously known methods.
  • the "antibody library” can subsequently be used for isolating polyclonal antibodies with a desired specificity. After having isolated specific polyclonal antibodies, the antibody library can be stored for later usage and the library can thus be "recycled" in an (in theory) indefinite number of times. It follows, that it is much faster to isolate antigen specific antibodies from an antibody library which in theory comprises antibodies that have antibody binding capability to almost any antigen - rather than immunising the animal and waiting several weeks for a specific antibody response.
  • a preferred way of obtaining an antibody library is by immunising with a peptide library obtainable by synthesizing peptides with a length of at least 6 amino acids.
  • the peptide antigen may also be a mix of peptides with different lengths.
  • the hen is ideal because it can produce about 1500 mg antibodies per month (about 50- 100 mg antibodies per egg (AOEA 24: 925-934, 1996).
  • antibodies can be obtained in a non-invasive manner from the chicken during the entire egg-laying period.
  • about 2-10% of the antibodies are antigen specific (AOEA 24: 925-934, 1996).
  • the percentage of antigen specific antibodies may be even higher as a response to immunisation with a multitude of antigens.
  • the antibodies are pooled and stored until a time where it is desired to isolate antigen specific antibodies.
  • Antibodies can be stored at subzero degrees (e.g. liquid nitrogen, -70 0 C, or -20 0 C).
  • the library may be preferable to avoid freezing by storing the library at temperatures in a range of about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 1O 0 C. Even though antibodies tend to be stable, repeated freeze/thaw circles may result in some degree of protein denaturation of the antibodies.
  • Antibodies according to the invention may furthermore be stored in the presence of one or more additives such as: antibiotics, anti-fungal compounds, antifreeze (e.g. glycerol), and buffer solutions.
  • a preferred additive is NaN 3 - preferably 0,1% NaN 3 in PBS.
  • Antibodies may even be stored at room temperature, preferably in the presence of preservatives, for relatively short period of times such as e.g. two weeks or less, preferably one week or less. This might be convenient e.g. in case of transport of antibodies via shipping, mailing, etc.
  • antibodies are collected from the animal after a period of at least 14 days, since this period of time is usually required to induce a detectable immune response in e.g. a chicken subsequent to immunisation.
  • the present invention furthermore relates to antibodies obtainable by the methods herein.
  • Another central aspect of the present invention relates to the use of antibody libraries being specific to a multitude of antigens.
  • the present invention thus relates to a method of isolating antigen specific antibodies, wherein the method comprises the following steps:
  • An antibody library obtained after immunising an animal with a peptide library generated by random synthesis thus provides a unique possibility of isolating antibodies with specificity to an antigen within a very short period of time - in theory within few hours.
  • a preferred solid phase is CnBr carrier.
  • a preferred coupling is Epoxy coupling since this approach is relatively cheap.
  • Ultrasound sonification or acidic conditions may e.g. be used for breaking the antigen: antibody binding in order to recover antigen specific antibodies.
  • various methods of coupling proteins to a carrier 10 and subsequently breaking protein: protein interactions are well known in the art.
  • the present invention relates to antibodies obtainable by the methods herein.
  • Peptide antigen Peptides according to the present invention are defined as peptides with a length of at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Peptides according to the present invention may also have lengths of about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300,
  • the peptides used as antigens according to the invention can be a mix of peptides of different lengths, i.e. there may be short peptides present with a length as short as 6 amino acids as well as longer peptides. Some or all of the peptides may also be in form of proteins being either denatured or in their naturally folded form. It follows that proteins can be
  • the peptides may in addition comprise any form of modifications such as e.g. glycosylation, lipid attachment, hydroxylation, disulfide bonding, phosphorylation, etc.
  • the peptides may also originate from a proteolytical digest of peptides and/or proteins.
  • Antigens according to the present invention are in other words of
  • the present invention does not relate to immunisation with antigens
  • 35 derived from cellular material including cell membranes, nucleic acids, etc.
  • the amount of different peptide antigens used for immunisation of the animal is at least 15.
  • the aim of the present invention is to provide tools for rapidly isolating antigen specific antibodies, it is advantageous to immunise with even more antigens, as the likelihood thus increases of obtaining antibodies with specificity to most antigens. It is therefore preferable to immunise with at least about 25, 50, 100, 150, 200, 250, 500, 1000, 5000, 10000, 50000, 100000, 500000, 1000000, or 5000000 different antigens. It is most preferable to immunise with a synthetically synthesized peptide library comprising, in theory, about 20 6 or more different peptide combinations.
  • Peptide library A preferred way of carrying out the present invention is to synthesize a peptide library with peptides of similar lengths covering the possible amino acid combinations of the 20 naturally occurring amino acids. Peptide synthesis may take place in solution or on a solid phase. A peptide library with peptides of a length of e.g. 6 naturally occurring amino acids will thus in theory have 20 6 different ways of combining the amino acids, and thus up to 20 6 different peptides.
  • the peptide library may also be a library comprising e.g. a specific fraction of the amino acid sequence from a number of different proteins with known sequences.
  • the peptides must have lengths of at least 6 amino acids in order to be able to bind to the hyper variable loops of the Fab fragment of the antibody. It follows that peptide libraries may also be prepared using less than 20 amino acids - in which case of course the theoretical number of different combinations will vary accordingly.
  • Carrier Conjugation to a carrier is important because peptides are small molecules that alone do not tend to be immunogenic, thus possibly eliciting a weak immune response.
  • the carrier contains many epitopes that stimulate T-helper cells, which help induce the B-cell response.
  • Many different carriers can be used for coupling to synthetic peptides. The most commonly selected carriers are keyhole limpet hemacyanin (KLH) and bovine serum albumin (BSA). The higher immunogenicity of KLH often makes it the preferred choice.
  • KLH keyhole limpet hemacyanin
  • BSA bovine serum albumin
  • Another advantage of choosing KLH over BSA is that BSA is used as a blocking agent in many experimental assays. Because antisera raised against peptides conjugated to BSA will also contain antibodies to BSA, false positives may result.
  • Ovalbumin is another useful carrier protein. It is a good choice as a second carrier protein when verifying whether antibodies are specific for the peptide alone and not the carrier.
  • Rabbit Serum Albumin may be used when the antibody response to the carrier protein must be kept to a minimum. Rabbits immunised with RSA conjugate are less likely to raise antibodies to the carrier, as the RSA is recognized as "self.” If the RSA conjugate were injected into another host, the protein would not be recognized as self. The same principle, of course, applies when immunising with chicken-derived carriers in chicken.
  • Suitable carriers will thus be of protein origin in most cases.
  • the carrier may be of non-protein origin such as e.g. polymers comprising carbohydrates, etc.
  • Peptides with a length of at least about 100 amino acids and more preferably with a length of at least about 150 amino acids may not need carrier conjugation in order to function as immunogens in the present invention. It is however advisable to conjugate all peptides with carrier molecules without regard to the size.
  • Immunisation of the animal may be carried out with or without pharmaceutical carriers.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
  • Immunisation of the animal may be carried out with adjuvants and/or pharmaceutical carriers.
  • An adjuvant is any substance that enhances the immune response to an antigen with which it is mixed. The antigen may also be mixed with two or more different adjuvants prior to immunisation.
  • Immunostimulatory oil emulsions for example, water-in-oil, oil-in-water, water-in-oil-in-water such as e.g. Freund's incomplete adjuvant such as Montainde®, Specol, mineral salts such e.g. as AI(OH) 3 , AIPO 4 , microbial products, Saponins such as Qual A, synthetic products, as well as adjuvant formulations, and immune stimulatory complexes (ISCOMs).
  • a list of other commonly used adjuvants is disclosed on pages 6-8 in WO 03089471, the list being hereby incorporated by reference.
  • the most preferred adjuvant according to the present invention is Specol since a fast immune response is initiated. Normally, antibodies can be harvested three to six weeks after immunisation in e.g. hen, but when using Specol as an adjuvant, antibodies may be harvested after only about two weeks.
  • Adjuvants are generally included in the immunogenic compositions in an amount according to the instructions of the manufacturer. If the adjuvant is Specol an amount of e.g. 5, 10, 15, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 ⁇ l may be added to the immunogenic composition. Preferably, the adjuvant is mixed with water or buffer in an amount of about 1 : 1.
  • Immunisation Antigen may be introduced into the animal by any suitable means. Preferably, the method of introduction involves injection. The animals may be immunised with the antigens intrasplenically, intravenously, intraperitoneally, intradermal ⁇ or subcutaneously or by any other suitable means. For practical and economical reasons, chickens kept under field conditions are vaccinated intramusculary in the breast muscle. In the laboratory, chickens can also be vaccinated subcutaneously in the neck.
  • the total number of immunisations will depend upon the type and dose of the antigen, as well as on the particular adjuvant employed. In a preferred embodiment, at least two immunisations should be given. If the antibody titres begin to decrease, booster immunisations can be given.
  • the total amount of peptide employed for each immunisation may likewise vary. Amounts of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 ⁇ g are preferably used in a single immunisation. But amounts of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 250, or 500 mg may also be used in a single immunisation.
  • Vertebrate animal A vertebrate animal is herein to be understood as an animal from the vertebrate phylae. Preferred animals are animals from either the mammalian or the avian orders. Avian animals are particularly preferred. Birds from the Galliformes order are most preferred. Birds used for immunisation should have an age of at least 17 weeks, preferably at least 19 weeks.
  • Antibodies are plasma proteins that bind specifically to particular molecules known as antigens. Antibody molecules are produced by B lymphocytes in response to immunisation with antigen. They are the specific molecules of the humoral immune response that bind to and neutralize pathogens or prepare them for uptake and destruction by phagocytes. Each antibody molecule has a unique structure that allows it to bind its specific antigen, but all antibodies have the same overall structure and are known collectively as immunoglobulins. The body can make an almost unlimited variety of different antibodies, each B lymphocyte being genetically programmed early in its development to produce antibodies of a single specificity. A pool of antibodies according to the present invention can be obtained by immunising a single animal with a randomly generated peptide library.
  • a randomly generated peptide library will in general stimulate an antibody response comprising antibodies with binding capability of (in theory) any antigen.
  • Antibody specificity toward an antigen According to the present invention an antibody can be termed antigen specific if e.g. antigen: antibody binding can be demonstrated in an assay as disclosed in Example 4, where antigen is linked to a carrier in the presence of PBS and antibodies are likewise suspended in a PBS buffer.
  • the antibodies obtained subsequent to immunisation show specificity to the antigens used for immunisation. If less than 1% of the antibodies show specificity to the antigens, antibodies may e.g. have been harvested too soon after immunisation, insufficient amounts of antigen have been employed, or inefficient amounts of adjuvants have been employed. More preferably, at least about 2, 3, 4, or 5% of the antibodies are antigens specific. Most preferably, at least about 6, 7, 8, 9, or 10% of the antibodies are specific. And most preferably about 20-30% of the antibodies are antigen specific.
  • Antigens are molecules that react with antibodies.
  • Elution agents that may be used include chaotropic agents such as guanidine hydrochloride or urea at concentrations between 10 ⁇ M and 8 M or ethylene glycol in an aqueous solution of 0.01% to 100% w/v. Elutions may also be carried out using aqueous or non-aqueous solutions of glycine. Elutions may be carried out using aqueous or non aqueous solutions of triethylamine between 1 ⁇ M and a saturated solution, preferably 100 mM, at a pH of between pH 8 and pH 13, preferably pH 11.5.
  • chaotropic agents such as guanidine hydrochloride or urea at concentrations between 10 ⁇ M and 8 M or ethylene glycol in an aqueous solution of 0.01% to 100% w/v. Elutions may also be carried out using aqueous or non-aqueous solutions of glycine. Elutions may be carried out using aqueous or non aqueous solutions of triethylamine
  • Galliformes The order of birds that includes grouse, ptarmigan, capercaillie, partridges, pheasants, quails, turkeys and peacocks. These are mainly grain-eating, heavy-bodied, ground-nesting birds, capable of only short, rapid flights. The cocks are usually more lively than the hens.
  • a preferred bird according to the present invention is a hen
  • Bursa fabricii A thymuslike lymphoid gland in birds that is an outgrowth of the cloaca and the site of B cell maturation.
  • a library of peptides with a length of 6 amino acids was synthesized using standard methods. The 20 naturally occurring amino acids were employed in each reaction and the library thus synthesized comprises (in theory) 20 6 different peptides.
  • the peptide sequence must contain at least one cysteine residue in order for the cross linking agent (MBS) to efficiently couple the peptides to the carrier protein Keyhole Limpet Hemocyanin (KLH).
  • MBS cross linking agent
  • KLH Keyhole Limpet Hemocyanin
  • a cysteine residue was added to the peptides as the seventh residue before carrier conjugation. It should be noted that it is also possible to conjugate via the amino terminal end when using MBS as a coupling agent.
  • Sephadex G-25 fine beads were swelled overnight in 5 ml buffer (0.1 M NaH 2 PO 4 pH 6.0)/g of dry beads at room temperature.
  • the swelled resin was washed 4 times using 0.1 M NaH 2 PO 4 pH 6.0 and poured into a 130 x 20 mm column. The column was subsequently equilibrated with 15 ml of 0.1 M NaH 2 PO 4 pH 6.0 and the resin was allowed to settle.
  • KLH 4 mg was dissolved in 0.3 ml of 0.1 M NaH 2 PO 4 pH 6.0. 1 mg m-Maleimidobenzoyl-N- Hydroxysuccinimide ester (MBS) in 150 ⁇ l of Dimethylformamide (DMF) was added drop- wise to the KLH solution under stirring. The KLH/DMF mixture was incubated at room temperature for 30 min on a rotating mixer.
  • MFS m-Maleimidobenzoyl-N- Hydroxysuccinimide ester
  • DMF Dimethylformamide
  • peak fractions at 280 nm pH 6.0 1 ml of peak fractions at 280 nm pH 6.0
  • 5 mg of the cysteine-containing peptide were mixed.
  • the peptide solution was mixed with the KLH-MBS conjugate and incubated for 12 to 16 hr at room temperature on a rotating mixing wheel.
  • the KLH-MBS-peptide conjugate was dialyzed for 24 hr at 4°C against 1 liter of PBS including two changes of buffer. Molecular weight cut off of the dialysis tube was 3,500.
  • composition used for each immunisation 10 ⁇ g KLH-peptide dissolved in 0.01 M NaCI buffer (pH 7.2) to a volume of 500 ⁇ l and 500 ⁇ l Specol to a total volume of 1 ml.
  • Yolk was separated from egg white.
  • the yolk was punctured and dissolved in 1: 10 weight/volume 2xH 2 O.
  • the mixture was frozen and thawed.
  • Ammonium sulfate was added to the thawed yolk to 25 % saturation, and the mixture was incubated at 20 0 C for 20 min.
  • the mixture was centrifuged in 30 min at 2500 x g.
  • the supernatant was harvested, ammonium sulfate was added to 40 % saturation, and the mixture was incubated at 20 0 C for 20 min.
  • the mixture was centrifuged in 30 min at 2500 x g.
  • Pellet comprising IgY was dissolved in 1 ml PBS/0.1% NaN 3 and kept at -20 0 C.
  • peptide 2.5 mg was dissolved in 0.5 M NaCI, 0.1 M NaHCO 3 , pH 8.3 and added to the swollen sepharose.
  • the peptide: resin mixture was mixed for 2 hours at room temperature and centrifuged at 40 x g for 5 min. The resin was subsequently incubated with blocking buffer (0.2 M Glycine, pH 8.0) at 4°C for 16 h.
  • the peptide: resin was then poured into a column and washed 5 x with 0.5 M NaCI, 0.1 M IMaHCO 3 , pH 8.3 followed by 5 ml 0.5 M IMaCI, 0.1 M ammonium acetate, pH 4.0.
  • Antigen specific antibodies were eluted from the column with 0.15 M NaCI, 0.2 M glycine, pH 2.2. The eluate was neutralized with 1 M Tris-HCL pH 8.0 and stored at 4°C with 0.01 % NaN 3 .
  • purified antibody is reloaded on column. Instead of eluting with pH 2.2 a range from pH 8.0 to pH 1.0 is used. The amount of antibody released at a specific pH is measured at 280 nm. The absorbents peak of the pH range 20 indicates its affinity.
  • Table 1 Induction of immune response in a hen to selected target antigens. The hen was immunised with a large amount of different peptides. Control samples were blood samples collected before immunisation. The results shown in the table are reproducible.

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Abstract

L'invention porte sur des procédés d'obtention d'anticorps polyclonaux chez des animaux, de préférence le poulet. Les méthodes de l'invention constituent des outils pour l'obtention d'anticorps spécifiques de l'antigène plus rapidement et de manière plus économique qu'avec les méthodes connues auparavant.
PCT/DK2006/050017 2005-05-03 2006-05-02 Methodes d'obtention d'anticorps specifiques de l'antigene Ceased WO2006117007A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
DK07722590.2T DK2027158T3 (da) 2006-05-02 2007-05-01 Metode til immunisering af en fugleart
PL07722590T PL2027158T3 (pl) 2006-05-02 2007-05-01 Sposób immunizacji gatunków ptaków
PCT/DK2007/000211 WO2007124755A1 (fr) 2006-05-02 2007-05-01 Méthode pour immuniser une espèce aviaire
EP11188731A EP2463306A1 (fr) 2006-05-02 2007-05-01 Procédé d'immunisation d'une espèce aviaire
EP07722590A EP2027158B1 (fr) 2006-05-02 2007-05-01 Méthode pour immuniser une espèce aviaire
US12/299,247 US8859233B2 (en) 2006-05-02 2007-05-01 Method for immunizing an avian species
DKBA201100184U DK201100184Y4 (da) 2006-05-02 2011-12-15 Antistoffer fra immuniserede fugle
US14/493,468 US9914766B2 (en) 2006-05-02 2014-09-23 Method for immunizing an avian species

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200500647 2005-05-03
DKPA200500647 2005-05-03

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/299,247 Continuation US8859233B2 (en) 2006-05-02 2007-05-01 Method for immunizing an avian species
PCT/DK2007/000211 Continuation WO2007124755A1 (fr) 2006-05-02 2007-05-01 Méthode pour immuniser une espèce aviaire

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007079755A1 (fr) * 2006-01-12 2007-07-19 Janus Beierholm Holding Aps Ré-immunisation et conception d'anticorps
US9914766B2 (en) 2006-05-02 2018-03-13 Carviar Aps Method for immunizing an avian species

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020168684A1 (en) * 1998-09-04 2002-11-14 Comb Michael J. Production of motif-specific and context-independent antibodies using peptide libraries as antigens
WO2003089471A1 (fr) * 2002-04-17 2003-10-30 European Molecular Biology Laboratory Procede de production d'anticorps monoclonaux

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020168684A1 (en) * 1998-09-04 2002-11-14 Comb Michael J. Production of motif-specific and context-independent antibodies using peptide libraries as antigens
WO2003089471A1 (fr) * 2002-04-17 2003-10-30 European Molecular Biology Laboratory Procede de production d'anticorps monoclonaux

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Title
COOK CARRIE L ET AL: "Simple purification methods for an alphagalactose-specific antibody from chicken eggs", JOURNAL OF BIOSCIENCE AND BIOENGINEERING, vol. 91, no. 3, March 2001 (2001-03-01), pages 305 - 310, XP002395979, ISSN: 1389-1723 *
HANLY ET AL.: "Review of polyclonal antibody production procedures in mammals and poultry", ILAR JOURNAL, vol. 37, 1995, pages 93-118, XP001247288 *
SCHADE ET AL.: "The production of avian (Eg yolk) antibodies: IgY", ATLA, vol. 24, 1996, pages 925-934, XP009071339 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007079755A1 (fr) * 2006-01-12 2007-07-19 Janus Beierholm Holding Aps Ré-immunisation et conception d'anticorps
US8859209B2 (en) 2006-01-12 2014-10-14 Carviar Aps Reimmunization and antibody design
US9938337B2 (en) 2006-01-12 2018-04-10 Carviar Aps Reimmunization and antibody design
US9914766B2 (en) 2006-05-02 2018-03-13 Carviar Aps Method for immunizing an avian species

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