Hen egg yolk antibodies

Akrum Hamdy

In 1893, Klemperer first demonstrated that the immunization of a hen resulted in the transfer of specific antibodies from the serum to the egg yolk.

For over a hundred years there was no scientific application for this knowledge .

But when the animal welfare became a matter of serious ethical concern for the scientific community, the results of Klemperer have attracted a great attention, particularly since the 1980s.

In the sense of animal welfare, the use of laying hens for antibody production represents a refinement and a reduction in animal use. It is a refinement in that the painful and invasive blood sampling or

scarifying are replaced by collecting eggs .The antibodies productivity in laying hens is nearly 20 times greater than that in rabbits (Schade et al., 2004).

Advanced Antibody platforms

Avian IgY antibodies

Recombinant antibodies

14 HAP Comprise ~96% of the Protein Mass in Plasma

Albumin

IgAIgG total

IgM

Pool Containing Medium- & Low-Abundance Proteins

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TransferrinFibrinogen

2-Macroglobulin

1-Antitrypsin

Haptoglobin

Apolipoproteins A-1 & A-II1-Acid Glycoprotein

SuperMix Approach – Dig Deeper

IgY12 Column

12 HAP-Depleted Fraction (Flow-Through of IgY12 Column: F1)

(Moderate/Low-Abundant Proteins, MAP/LAP)

Protein Mixture as Antigens for

IgY Production

Antigen- Affinity Column

IgY against 12 HAP-Depleted Fraction

(SuperMix)

Eluted Fraction (E1) (12 High-Abundant

Proteins – HAP)

Eluted Fraction (E2) (MAP or Immunoreactive Proteins)

Flow-Through Fraction (F2) (LAP or Non-Immunoreactive Proteins)

SuperMixColumnPlasma Directly Applied to IgY-SuperMix Column

Plasma(P)

IgYs

IgY-Immunoaffinity Separation

1. High avidity of binding

2. Specific removal of the targets

3. Sample size 20-500 l

4. Recyclable >100x

5. Efficacy >95%

6. Minimal loss of non-targets

IgY-BEADS’ FEATURES

Why Chickens & IgYs?

Hen Rabbit

Distance toHuman -205 Myr +72 Myr

Production Accumul Why Chickens & IgYs? ativeInvasive

Monthly 20 Eggs 50 ml Blood

Ab Quantity 100 mg/egg 10 mg/ml

Maintenance Low High

Unit Cost Low High

AffinityMaturation

GeneConversion

Avidity High Good

Somatic Mutation

SepproTip Design

Membrane filter

Stainless steel ring500 µl

SepproTip IgY-12

IgY Microbeads (Seppro® IgY-12)

Membrane filter

SepproTip Process

500 µl Unfractionated Sample (1:33

Dilution)

Binding Washing Eluting Neutralizing Regenerating

Pipetting Up and Down 8 Times Each

Pipetting Up and Down 8 Times Each

Up and Down 10 Times

Blood Samples

Loading 15 µl

Option to Collect the Washes at

Position 2

Collect and Pool Elution Fractions at Positions 3 and 4

Pipetting Up and Down 15 Minutes

Position 1 Wells 1-3 Wells 4-7 Well 8 Wells 9-10

Up and Down 3 Times

Liquid in and stop

SepproTip Content - Customizable

MediaPurposeCapacity

IgY12Partitioning of Top-12 highly-abundant

proteins from human and other primate serum or plasma

15 µl of serum/plasma

IgY-R7Partitioning of Top-7 highly-abundant

proteins from rat serum or plasma10 µl of

serum/plasma

IgY-M7Partitioning of Top-7 highly-abundant

proteins from mouse serum or plasma.10 µl of

serum/plasma

IgY-D11Partitioning of Top-11 highly-abundant

proteins from dog serum or plasma.10 µl of

serum/plasma

SuperEnrich

Enrichment of tissue leakage proteins, cell line-specific

500 ul of plasma/serum

Anti-Rubisco

Removal of Rubisco protein from plant extract (plant biomarker discovery)

0.8 mg of Rubisco

IgY: Less Non-Specific Binding

Mammalian IgG

Chicken IgY

Complement

Rheumatoid Factor

IgM

Fc Receptor

No Binding to Fc Region

Polyclonal Antibody Production

Total IgYs

Bioinformatic Analysis

Expression Vectors

Antigens Ligands

Affinity Purification Quality Control Tests

Chickens and Eggs

Protein Expression Contract Services

•Bioinformatics Analysis

System Selection and Adaptation

Refolding and Solubilization

Protein Expression and Optimization

Scale-Up Production

GLP/cGMP Compliance

Vector Design and Construction

Comparison of IgY and IgG

[35S] LabelingECL Detection

Western Blot Immuno-precipitation

Immuno-staining

IgY IgG

A B C

IgY IgG

Calnuc

Anti-Calnuc IgY(Golgi-Specific)

Body’s defenses against bacteria

Neutralizing antibodies: block viruses from attaching and entering the host

Virus specific antibodies: can act against proteins on the virus surface

Overuse of antibodies can lead to the development of resistant bacteria

conclusion

With the ban on sub-therapeutic antibiotic usage in Europe and the increasingly strictness of the European legislation on food hygiene, passive immunization by oral administration of pathogen-specific hen egg yolk antibody (IgY) may be a

useful and attractive alternative .Furthermore, because of the high yolk antibodies concentration, over 100 mg of antibodies can be obtained from one egg (Akita et al., 1992). Since a laying hen produces approximately 25 eggs per month, 5 g of antibody per month may be obtained from a single laying hen.