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 N NaOH=14.4; in 5M guanidine=12.7
(Leslie and Clem 1969)
- weight of IgY (or other protein) to molar quantity can be converted here
Name IgY has been 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?
Affinity constant for polyclonal antibodies cannot be really measure, since they bind not 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.
Bidning 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 to how many places the antibody can bind antigen. IgY can perform, as IgG, bi or monovalent, depending upon the size of the antigen. Therefore the binding strength of polyclonal antibody can be high, although the affinity is low.
One can often meet statements that chicken antibodies have low affinity to the antigens.
However, especially, when antigens are more foreign to the hens than to the rabbits, produced antibodies can have higher multivalency in the chicken, compare to the rabbit, even though the affinity can be lower.
In mammals the Ab diversity is mainly performed by rearranging various gene segments to produce the hypervariable part of the antibody and additionally by somatic mutations. In this way the production of more than a million Ab specificities is possible. In hens the Ab diversity is mainly achieved by gene conversion and in addition by V-J flexible joining and by somatic point mutation as in mammals. In contrast to mammals, in the hens there is only 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 of 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, in large amounts passed to egg yolk and 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 (Horton et al. 1984)
- preferably at pH8 the Fc part of IgY is firmly bound to the latex particles (Dávalos-Pantoja and Hidalgo-Alvarez, 2001)
12 eggs contain around 1 gram of total IgY antibodies, what is equivalent to 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/month. Amount of antigen-specific antibodies varies from 0,5-10% of total IgY, depending upon 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 (called also 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 fraction is very stable. Even at room temperature (although we do not recommend it as storage conditions). IgY can be stored at + 4°C with 0.02 % sodium azide (note: azide inhibits activity HRP enzyme) or gentamicin sulfate (50 ug/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 ug/ml). Egg yolk should NEVER be frozen.
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; Final concentration of glycerol 10 or 50%
- -20°C with BSA at final concentration of 0.05-0.5%
- For larger volumes of affinity purified antibodies, filter-sterilise antibody sample and aliquot to avoid multiple freezing and thawing.
- Storage at protein concentration around 0.5-1 mg/ml.
Alternative agents for preventing bactrial growth in antibody solution:
- Thimerosal at 0,01%
- Gentamicin sulfate at 50 ug/ml