IgY - main low molecular weight immunoglobulin present in hen's serum and egg yolk in concentration of around 5-20 mg/ml
Molecular mass [kDa] ~ 180 (light chain ~ 25 [kDa] each; heavy chain ~ 65-68 [kDa] each)
- Isoelectric point 5.7 - 7.6 (6.6 +/- 0.9 Davalos-Patoja et al. 2000)
- The most hydrophobic moiety of IgY molecule is the Fc fragment (Schade et al. 2005)
- Extinction coefficient (i.e. absorbance of a 10 mg/ml solution at 280 nm) are: in 0.3 M KCl = 13.18; in 0.1 M NaOH = 14.4; in 5 M guanidine = 12.7
(Leslie and Clem 1969)
- Weight of IgY (or other protein) to molar quantity can be converted here
The name IgY was proposed by Leslie and Clem in 1969. The authors showed experimental data proving that IgY molecule is different from IgG ("Phylogeny of immunoglobulin structure and function" G. A. Leslie and L.W. Clem, Journal of Experimental Medicine, Vol. 130, No. 6: 1337-1352, 1969). Other names for IgY (often misleading) met in literature are: Chicken IgG, Egg Yolk IgG, 7SIgG.
What affinities one can expect from IgY antibodies in comparison with IgG antibodies?
The affinity constant for polyclonal antibodies cannot properly be measured, since they do not bind to one epitope only. Affinity describes a single antibody-antigen interaction, between one antigen binding site and one epitope on the antigen, and can be measured for monoclonal antibodies.
Binding strength of polyclonal antibodies can be described as avidity, which is a combination of the affinities to the individual binding sites and the valency of the antibody. Valency describes the number of sites that the antibody can bind to the antigen. IgY, as IgG, can be bi or monovalent, depending on the size of the antigen. Therefore, the binding strength of a polyclonal antibody can be high, even though the affinity is low.
A common misconception is that chicken antibodies have low affinity to antigens. However, antibodies produced in chicken can have higher multivalency, compared to rabbit antibodies, even though the affinity may be lower. This is especially true when the antigens are more foreign to the chicken than rabbit.
In mammals the Ab diversity is mainly achieved by rearranging various gene segments to produce the hypervariable part of the antibody, and additionally by somatic mutations. This way, the production of more than a million Ab specificities is possible. In hens, the Ab diversity is mainly achieved by gene conversion, V-J flexible joining, as well as by somatic point mutation as in mammals. In contrast to mammals, hens only have one functional VH- or VL-gene, but in addition there are approximately 25 of the so-called pseudo-V genes (lacking the usual transcription regulatory and signal-recognition sequence). Therefore, hens can often produce antibodies which will recognize different epitopes than mammalian antibodies would do.
Some interesting features of IgY:
- Does not bind to rheumatoid factor (an inflammatory response marker) in blood (Larsson et al. 1988)
- Does not activate mammalian complement factors (Larsson et al. 1992). This can be a great advantage in case of assay development for mammalian serum samples
- Does not bind to cell surface Fc receptor (Schmidt et al. 1993)
- Does not bind to protein A (Kronvall et al. 1974) or protein G (Akerström et al. 1985)
- Latex particles sensitized by IgY molecules do not aggregate by means of the rheumatoid factor (as is the case of IgG antibodies). Moreover, IgY-latex complexes have higher colloidal stability than IgG at pH 8 (L. Davalos-Pantoja et al. 2000)
- IgY antibodies are selectively, and in large amounts passed to egg yolk. Therefore, no IgM and IgA impurities can be found in IgY preparations (Schade et al. 2001)
- Might bind three to five times more secondary antibody than IgG (Horton et al. 1984)
- At pH 8, the Fc part of IgY is firmly bound to the latex particles (Dávalos-Pantoja and Hidalgo-Alvarez, 2001)
12 eggs contain around 1 g of total IgY antibodies, which is equivalent to the amount of total IgG antibodies present in around 100 ml of serum.
A single hen can substitute up to 12 rabbits in antibody production over one year. Around 2.5 g of total IgY antibodies can be produced per hen and month. The amount of antigen-specific antibodies varies from 0.5-10% of total IgY, depending on the antigen used.
Recommended manual about IgY:
"Chicken Egg Yolk Antibodies, Production and Application" R. Schade et al., 2000, Springer-Verlag, Lab Manuals, ISBN 3-540-66679-6.
Video tutorial of IgY purification.
Secondary antibodies to chicken IgY (also called chicken IgG), quality and price competitive
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How to store IgY antibodies?
Total IgY fraction (IgY antibodies purified by precipitation from egg yolk)
Purified IgY fractions are very stable, even at room temperature (although we do not recommend these storage conditions). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml).
Avoid freezing and thawing of IgY or storing it on dry ice. IgY antibodies can be stored at -20°C.
"The IgY preparations were stable over time. No loss of antigen recognition was observed after storage for 3 years at + 4°C". F. De Ceunick et al. Journal of Immunological Methods 252 (2001) 153-161.
Antibodies in egg yolk should be stored at 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 µg/ml). Egg yolk should NEVER be frozen.
Affinity purified antibodies
Affinity purified antibodies are the most fragile. Care should be taken when considering storing conditions, which should be checked experimentally for every single antibody. Affinity purified antibodies against different epitopes can vary in stability. Some will precipitate directly after the purification, while the activity may still remain. It is difficult to predict storage conditions for a given antibody in advance - there are some alternatives to be tested:
- -20°C or -80°C
- + 4°C with preservatives like azide (0.02%) or merthiolate
- -20°C with glycerol at a final concentration of 10% or 50%
- -20°C with BSA at a final concentration of 0.05-0.5%
- For larger volumes of affinity purified antibodies, filter-sterilize the antibody sample and aliquot to avoid repeated freezing and thawing.
- Storage at protein concentration around 0.5-1 mg/ml.
Alternative agents for preventing bacterial growth in antibody solution:
- Thimerosal at 0.01%
- Gentamicin sulfate at 50 µg/ml